Fragment Based Identification of Phosphatase Inhibitors ... · Inhibitor library synthesis and...
Transcript of Fragment Based Identification of Phosphatase Inhibitors ... · Inhibitor library synthesis and...
Fragment Based Identification of Phosphatase Inhibitors
by
Tyler Daniel Baguley
A dissertation submitted in partial satisfaction of the
requirements for the degree of
Doctor of Philosophy
in
Chemistry
in the
Graduate Division
of the
University of California, Berkeley
Committee in charge:
Professor Jonathan A. Ellman, Co-Chair
Professor Ming C. Hammond, Co-Chair
Professor Matthew B. Francis
Professor Jasper Rine
Fall 2014
© Copyright by
Tyler Daniel Baguley
2014
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Abstract
Fragment Based Identification of Phosphatase Inhibitors
by
Tyler Daniel Baguley
Doctor of Philosophy in Chemistry
University of California, Berkeley
Professor Jonathan A. Ellman, Co-Chair
Professor Ming C. Hammond, Co-Chair
Chapter 1. In this chapter, phosphatases are broadly introduced as an enzyme class with
therapeutic implications in a variety of disease areas. Different methods for inhibitor
identification are outlined as well. Finally, a substrate-based fragment approach which has been
developed by the Ellman group for the identification of phosphatase inhibitors is introduced.
Chapter 2. The fragment-based method for the identification of phosphatase inhibitors
introduced in chapter 1 is applied to Mycobacterium tuberculosis protein tyrosine phosphatase
PtpA. Inhibitors incorporating a well established phosphate mimic, the
difluoromethylenephosphonic acid, were explored, resulting in low micromolar inhibitors of
PtpA. The most potent compound was also shown to be selective for PtpA over a variety of
human phosphatases as well as Mycobacterium tuberculosis protein tyrosine phosphatase PtpB.
This inhibitor represents a chemical tool that can be used in conjugation with PtpB selective
inhibitors described previously within the Ellman group to further probe the roles of PtpA and
PtpB in tuberculosis infection.
Chapter 3. The fragment-based approach introduced in chapter 1 is applied to striatal-
enriched protein tyrosine phosphatase (STEP), a brain specific phosphatase that has been
implicated in a number of neuropsychiatric disorders such as Alzheimer’s disease. STEP is a
very promising target for these diseases and was discovered nearly 20 years ago, yet no small
molecule inhibitor existed prior to our work. Through our fragment-based approach, we were
able to identify many low molecular weight (<450 Da), nonpeptidic, single-digit micromolar
mechanism-based STEP inhibitors with greater than 20-fold selectivity across multiple tyrosine
and dual specificity phosphatases. Additionally, significant levels of STEP inhibition in rat
cortical neurons were also observed.
Chapter 4. This chapter discusses the discovery and characterization of benzopentathipins as
redox-reversible inhibitors of STEP, the therapeutically relevant phosphatase introduced in
2
chapter 3. The majority of the chapter focuses on the biochemical characterization of the
benzopentathiepin 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6 amine hydrochloride (TC-
2153), a unique compound with a cyclic polysulfide that forms a reversible covalent bond with
the catalytic cysteine in STEP. Several analogs of TC-2153 are prepared to scope out not only
what is important for inhibition, but also to identify locations on the molecule that are amenable
to diversification for further compound development. Importantly, TC-2153 is shown to be
active in cell-based secondary assays and in animal behavioral models.
Chapter 5. This chapter outlines the use of seleninic acids as redox-reversible inhibitors of
STEP. The redox-reversible mode of inhibition described in chapter 4 is adapted to seleninic
acids, which have been demonstrated to form stable S–Se bonds with cysteine thiols. This new
PTP pharmacophore is merged with the SAR determined in chapter 3 to attain an inhibitor with
good activity in vitro.
Chapter 6. The development of additions of Knochel-type benzyl zinc reagents to N-tert-
butanesulfinyl aldimines is described. These additions utilize sp3-hybridized reagents that show
good functional group compatability, adding chemoselectively to imines that possess ester and
nitrile functionality. Addition to a glyceraldehyde-derived imine proceeds in high yield and
excellent selectivity and provides entry to hydroxyethylamine-based aspartyl protease inhibitors.
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Table of Contents
Chapter 1. Protein tyrosine phosphatases as therapeutic targets 1
Protein tyrosine phosphatases 2
Redox-reversible regulation of phosphatases 3
Methods for identifying phosphatase inhibitors 3
High throughput screening 3
X-ray and NMR fragment screens 4
Substrate activity screening 4
Enzymatic assays 5
Substrate screening assay 5
Inhibitor screening assay 6
References 7
Chapter 2. Identification of inhibitors of the Mycobacterium tuberculosis
phosphatase PtpA 9
Introduction 10
Initial scaffold identification 10
Conversion to inhibitors 11
Inhibitor library synthesis and evaluation 11
Amide replacement analog synthesis and evaluation 12
Benzanilide scaffold optimization and evaluation 15
Inhibitor selectivity profile 17
Modeling studies 18
Conclusions 18
Experimental 19
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References 31
Chapter 3. Fragment-based identification of inhibitors of striatal-enriched
protein tyrosine phosphatase 35
Introduction 36
Inhibitor scaffold identification 36
Optimization of inhibitor 3.15 39
Optimization of inhibitor 3.16 42
Inhibitor selectivity profile 46
STEP inhibition in neuronal cultures 46
Blood-brain barrier permeability 46
Conclusions 47
Experimental 48
References 77
Chapter 4. Benzopentathiepins as novel redox-reversible inhibitors of STEP 81
Introduction 82
STEP as a therapeutic target 82
Initial high throughput screening results 82
Benzopentathiepins as attractive target molecules 84
Synthesis of TC-2153 84
Mechanism of STEP inhibition by TC-2153 86
Enzymatic characterization of inhibition 86
LC-MS/MS characterization of inhibition 87
Preparation of TC-2153 analogs for STEP inhibition 88
TC-2153 analog synthesis 89
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Inhibition of STEP by TC-2153 analogs 90
TC-2153 activity in cell-based secondary assays and in vivo 92
TC-2153 activity in cortical neurons and in vivo 92
TC-2153 specificity in vivo 92
TC-2153 reduces cognitive deficits in 3xTg-AD mice 94
Conclusions 95
Experimental 96
References 108
Chapter 5. Seleninic acids as redox-reversible inhibitors of STEP 111
Introduction 112
Synthesis of seleninic acid inhibitors 113
In vitro evaluation of inhibitors 114
Conclusions 115
Experimental 116
References 122
Chapter 6. Asymmetric additions of Knochel-type benzyl zinc reagents to N-
tert-butanesulfinyl aldimines 123
Introduction 124
Optimization of benzyl zinc additions 125
Evaluation of substrate scope for diastereoselective benzyl zinc addition 126
Stereochemical rationale 126
Preparation of aspartyl protease inhibitor precursors 128
Conclusions 129
Experimental 129
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References 141
Appendix 6.1: X-ray crystal data for compound 6.27 145
Appendix 6.2: X-ray crystal data for compound 6.31 157
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Table of Abbreviations
%R membrane retention (for PAMPA)
[]20
D specific rotation at the sodium D line at 20 °C
[I] inhibitor concentration
[S] substrate concentration
°C degrees Celsius
3xTg-AD triple transgenic Alzheimer’s disease mice
Å angstrom
ABq AB quartet
Abs absorbance
Absmax wavelength of maximum absorbance
Ac acetyl
AcCl acetyl chloride
acetone-d6 deuterated acetone
AcOH acetic acid
AD Alzheimer's disease or 3xTg-AD mouse
AD-TC 3xTg-AD mouse treated with TC-2153
AD-Veh 3xTg-AD mouse treated with vehicle control
AEBSF 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride
ANOVA analysis of variance
app apparent
Ar aryl
Asp aspartic acid
avg average
BBB blood-brain barrier
Bn benzyl
BnBr benzyl bromide
Boc tert-butyloxycarbonyl
Boc2O di-tert-butyl dicarbonate
br broad
br s broad singlet
br t broad triplet
c concentration in grams per deciliter (g/dL)
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calcd. calculated
CD3OD dueterated methanol
CN control
cod 1,5-cyclooctadiene
cpd compound
Cys cysteine
NMR chemical shift, in ppm
d doublet
dr diastereomeric ratio
Da dalton
dd double of doublets
dec decomposition
DFMP difluoromethylenephosphonic acid
DIAD diisopropyl azodicarboxylate
DiFMUP 6,8-difluoro-4-methylumbelliferyl phosphate
DM double mutant (3xTg-AD + STEP–/–
)
DMA N,N-dimethylacetamide
DMDO dimethyldioxirane
DME 1,2-Dimethoxyethane
DMF N,N-dimethylformamide
DMSO dimethyl sulfoxide
DMSO-d6 deuterated dimethyl sulfoxide
dppf 1,1′-bis(diphenylphosphino)ferrocene
dt doublet of triplets
dtbpy 4,4-di-tert-butyl bipyridine
DTT dithiothreitol
DUSP dual-specificity protein tyrosine phosphatase
EDTA ethylenediaminetetraacetic acid
ee enantiomeric excess
equiv equivalents
ERK1/2 extracellular signal-regulated kinases 1 and 2
ESI electrospray ionization
Et ethyl
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Et2O diethyl ether
Et2SiH2 diethylsilane
Et3SiH triethylsilane
EtOAc ethyl acetate
EtOH ethanol
FAB fast atom bombardment
g gram
GAPDH glyceraldehyde 3-phosphate dehydrogenase
GC gas chromatography
GCMS gas chromatography-mass spectrometry
GluN2B subunit of the NMDAR
GSH reduced glutathione
GSSG oxidized glutathione dimer
GST glutathione S-transferase fusion tag
h hour
HBpin pinacolborane
hept heptet
HPLC high performance liquid chromatography
HRMS high resolution mass spectrometry
HTS high throughput screening
Hz hertz
i.p. intraperitoneal
IC50 half maximal inhibitory concentration
iPr isopropyl
iPr2NH N,N-diisopropylamine
iPrOH isopropanol
IR infrared spectroscopy
J NMR coupling constant
JCF NMR coupling constant between 13
C and 19
F atoms
JPF NMR coupling constant between 31
P and 19
F atoms
KHF2 potassium hydrogen difluoride
Ki the dissociation constant for an enzyme and inhibitor
kinact rate constant of inactivation
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Km the Michaelis constant, the dissociation constant for an enzyme and substrate
KO genetic knockout
KOAc potassium acetate
kobs observed rate constant
L liter
wavelength
LCMS liquid chromatography-mass spectrometry
LD50 median lethal dose
LDDN Laboratory for Drug Discovery in Neurodegeneration
LMW-PTP low molecular weight protein tyrosine phosphatase
Ln ligand
micro
M molar
m multiplet
m.p. melting point
m/z mass-to-charge ratio
Me methyl
MeOH methanol
MES 2-(N-morpholino)ethansulfonic acid
MESG 2-amino-6-mercapto-7-methylpurine riboside
mg milligram
mg/mL milligrams per milliliter
MHz megahertz
MIB 3-exo-(morpholino)isoborneol
min minute
mL milliliter
L microliter
M micromolar
m micrometer
mmol millimole
Mnt menthyl
mol mole
Ms mesyl, methanesulfonyl
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Mtb Mycobacterium tuberculosis
MTBE methyl tert-butyl ether
MWM Morris water maze
N normal, equivalent concentration
Na3VO4 sodium orthovanadate
NADPH nicotinamide adenine dinucleotide phosphate
NaSH sodium hydrosulfide
NBS N-bromosuccinimide
n-BuLi n-butyllithium
nd not determined
nM nanomolar
nm nanometer
NMDA N-methyl-D-aspartate
NMDAR N-methyl-D-aspartate receptor
NMR nuclear magnetic resonance or nuclear magnetic resonance sprectroscopy
OD600 optical density at 600 nm
p pentet
p probability-value, p-value
PAMPA parallel artificial membrane permeability
Pd(PPh3)4 Tetrakis(triphenylphosphine)palladium(0)
Pe effective permeability (for PAMPA)
Ph phenyl
PK pharmacokinetics
PNP purine nucleoside phosphorylase
pNPP p-nitrophenyl phosphate
PPh3 triphenylphosphine
ppm parts per million
p-protein phosphoprotein
ps picosecond
pSer phosphoserine
pThr phosphothreonine
PTP protein tyrosine phosphatase
pTyr phosphotyrosine
x
Pyk2 proline-rich tyrosine kinase 2
q quartet
RIPA radioimmunoprecipitation assay
ROS reactive oxygen species
rt room/ambient temperature
s singlet or second
S.D. standard deviation
s.e.m. standard error of the mean
S8 or S8 elemental sulfur
SAR structure activity relationship
SAS substrate activity screening
Ser serine
sext sextet
SNAr nucleophilic aromatic substitution
SOD superoxide dismutase
STEP striatal-enriched protein tyrosine phosphatase
STEP–/–
STEP genetic knockout
t triplet
TAT transactivator of transcription fusion tag
TB tuberculosis
TBAF tetra-n-butylammonium fluoride
TBS tert-butyldimethylsilyl
TBSCl tert-butyldimethylsilyl chloride
tBu tert-butyl
TC TC-2153 treated
TC-2153 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride
td triplet of doublets
TEEDA tetraethylethylenediamine
TFA trifluroacetic acid
TFAA trifluroacetic anhydride
THF tetrahydrofuran
Thr threonine
TMS tetramethylsilane
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TMSCl chlorotrimethylsilane
TMSI iodotrimethylsilane
tr,major retention time, major component
tr,minor retention time, minor component
tris 2-amino-2-hydroxymethyl-propane-1,3-diol
Trp tryptophan
U units of enzyme activity
U/mL units of enzyme activity per milliliter
UPLC ultra performance liquid chromatography
UV ultraviolet
UV-Vis ultraviolet-visible absorption spectroscopy
Veh vehicle control
Vmax maximum reaction velocity
Woollins' reagent 2,4-Diphenyl-1,3,2,4-diselenadiphosphetan-2,4-diselenide
WT wild-type
WT-TC wild-type mouse treated with TC-2153
WT-Veh wild-type mouse treated with vehicle control
Y tyrosine
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Acknowledgements
First and foremost, I need to thank my advisor professor Jonathan Ellman. His guidance,
insight and advice, coupled with his overall knowledge and love of chemistry, have been
invaluable. He has inspired me to be a better scientist and has pushed me at every step along the
way. Working for Jon has been one of the most rewarding aspects of my PhD. He has struck the
perfect balance of openness and willingness to help at every step and the ability to step back and
let the students drive the science which has been very challenging at times, yet rewarding.
I’d also like the thank professor Richmond Sarpong at UC Berkeley. He allowed me as an
incoming graduate student to work in his group prior to enrollment at Berkeley. His group was
the first serious research environment I was part of, and they taught me many of the basics of
how to be a better scientist, as well as the simple things like how to properly run a column. After
the move to Yale, I had the wonderful opportunity to team up with researchers at the Yale School
of Medicine, and I would be remiss not to mention professors Paul Lombroso and Angus Nairn.
Their collaboration through the last few years of my PhD has been invaluable and extremely
rewarding. Paul’s students Jian Xu and Manavi Chatterjee have been the perfect collaborators;
open, helpful, informative, efficient, and overall great scientists.
I am indebted to the Ellman group as a whole, past and present, Berkeley and Yale, graduate
students, postdocs and undergrads alike. You have helped me survive the past 5+ years and
shaped me into the scientist I am today. When I started at Berkeley, my labmates in 908 Latimer
were extremely helpful with the day to day questions, as well as great friends. Denise Colby,
Melissa Herbage, MaryAnn Robak and Melissa Leyva were the ideal labmates to begin my
career. Whether it was annoying Denise or Melissa with trivial science or Leyva about sports
(Go Ducks!) there was never a dull moment in lab.
Rhia Martin, Pete Marsden, Andy Tsai and MaryAnn were always up to go to Yogurt Park or
Chipotle, and to hang out outside of lab. I was sad to leave some of you behind at Berkeley,
because I knew I would miss our weekend meals and overall hanging out (I don’t miss step
aerobics though). I’d also like to thank Katherine Rawls for being my first, and only, true mentor
in the lab. It was great to join in on established projects and you were invaluable to my
development as a scientist. I will always remember Morten Storgaard, Van Yotphan, Jason Ji and
Vivian Lin for their unique laughs and smiles. I’m sad Jason and Vivian didn’t make the trek to
New Haven, but I can’t say I blame them.
Kyle Kimmel, Somenath Chowdhury, Andrew Buesking, Rhia and Andy all made the move
to Yale more bearable, whether it was Rhia’s laugh, skiing with Andy or Somenath’s sense of
humor (I think), I always had a good time and couldn’t have asked for a better group with which
to make the transition across the country. Once at Yale, I had the wonderful opportunity to work
along many talented chemists in 321 CRB. Both Yajing Lian and Kevin Hesp were excellent
(and productive) chemists, and I am glad to have worked alongside both of them. I leave the bay
in capable hands, although I hope Jessica Yuan gets some companions soon.
Although not in my bay, I will remember Tatjana Huber’s smile and Jimmie Weaver’s
breadth of knowledge (not to mention his coffee intake). Eric Phillips had a no nonsense
approach to chemistry which is admirable in its own right, and Simon Duttwyler continued that
tradition once Eric left, essentially training many of the current members of the group on how to
be rigorous with or without a glovebox. Michael Ischay’s ability to be both an optimist and
pessimist at the same time still confuses me, but he knew how to be a scientist, depending on
which day you asked him. Haichao Xu and Joey Stringer were fantastic collaborators, even if I
hardly saw them as they were stuck out at West Campus.
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To the current members of the group, keep on trucking. Hasan KHAAANN (or is it Haroon
Kzing?), Haya (brown Kate) Jamali, Kate (white Haya) Otley and Caroline Tjin should be more
than enough to keep the chemical biology projects rolling. You all are great chemists and
fantastic people (even if you are Canadian, Hasan). Josh, eventually you’ll have a project where
the products are simple to separate, but until then, keep up the good work. Thanks to James
Phelan for, well, everything from hanging out outside of lab to answering my silly questions
which I should know the answer to after five years. Tehetena Mesganaw, it will be a pleasure to
see you back on the best coast shortly. Shuming Chen’s knowledge of computational chemistry
has been an asset to the group and has expanded my understanding of the subject. I have enjoyed
my college football discussions with Apiwat Wangweerawong, and maybe this year the Noles
and the Ducks can meet in the championship game to settle it once and for all (wishful thinking).
To the younger chemists in the group, Jeff Boerth and Scott Kolmar, keep up the good work and
progress will come, I promise. Finally, I’d like to thank Andrew “Wayne” Buesking for being
one of my best friends in the Ellman group over my whole time here. We made the trek from
Berkeley together and now, we’ve made it to the end. I’d also like to thank Andrew’s wife
Melissa for having us over so often, even that time when we invited ourselves when it was
snowing. (If I never live somewhere where snowplows are needed again, it will be too soon).
Finally, I’d like to thank my friends and family who have helped me either directly or
indirectly to pursue this degree in chemistry. First, I’d like to thank my parents, Dan and Tina,
for being supportive every step of the way. I’d like to thank my siblings Jason and Kristen for
both picking holiday weekends to get married, as I love paying double rates for cross-country
flights. I’d like to thank the many math and science teachers that inspired me throughout the
years to pursue knowledge and not grades: Mr. Miller, Mr. Sparkman in high school and Gautam
Bhattacharyya at the University of Oregon to name a few. I’d also like to thank Professor John
Keana at the University of Oregon for giving me a chance as an undergraduate to do chemical
research and really whet my appetite for science.
Last, but certainly not least, I must thank my wonderful wife, friend, partner and (at times)
colleague, Stephanie, without whom graduate school would have been a lot less pleasant. She
has listened to my frustrations in spite of her own, been my first source of editing on all my
manuscripts so I didn’t send something stupid to Jon, and has even helped me with biology
techniques and materials at times. She’s picked up my slack around the house, even though she
has her own job and responsibilities, and made sure I didn’t eat at the carts every day for lunch.
Even though I “dragged her ass to Connecticut,” she’s done it all (mostly) with a smile, and has
only had one psychotic break. I truly have appreciated all her support and understanding.
xiv
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Chapter 1. Protein tyrosine phosphatases as therapeutic targets
Abstract: This chapter serves as the introduction to my doctoral thesis. Protein tyrosine
phosphatases (PTPs) are introduced as an enzyme class that catalyzes the hydrolysis of
phosphate groups from phosphorylated tyrosine residues in proteins. Additionally, multiple
methods to attain PTP inhibitors which have been utilized with varying degrees of success are
introduced. Finally, substrate activity screening (SAS), a substrate-based fragment identification
approach developed within the Ellman group prior to my arrival, is outlined, including an
introduction to the enzymatic assays performed throughout the thesis.
2
Protein tyrosine phosphatases
Phosphatases are enzymes that catalyze the dephosphorylation of proteins and other
molecules in living organisms. They work in concert with kinases, which catalyze the
phosphorylation of these biomolecules, to control a variety of cellular processes, from cell
growth and differentiation to immune response.1 There are two major classes of protein
phosphatases: protein tyrosine phosphatases (PTPs), which primarily dephosphorylate
phosphotyrosine (pTyr) residues, and the Ser/Thr phosphatases, which target phosphoserine
(pSer) and phosphothreonine (pThr). The PTP superfamily can be further separated into three
subfamilies: classical PTPs, which dephosphorylate only pTyr; dual-specificity PTPs (DUSPs),
which can accommodate pTyr, pSer and pThr; and low molecular weight PTPs (LMW-PTPs),
which share no significant homology with either classical PTPs or DUSPs, but also only
accommodate pTyr residues.2 While Ser/Thr phosphatases are metalloenzymes, all known PTPs
catalyze dephosphorylation through the formation of a covalent phosphocysteine intermediate
followed by subsequent hydrolysis by a solvent water molecule (Figure 1.1).3
Figure 1.1. Catalytic mechanism of protein tyrosine phosphatases (PTPs).
Aberrant PTP activity has been associated with many diseases, including diabetes (PTP1B),
cancer (Cdc25), autoimmune disease (CD45) and neurodegenerative disease (STEP).
Additionally, phosphatases have also been recognized as virulence factors for many infectious
diseases, such as bubonic plague (Yersinia PTP), tuberculosis (Mycobacterium tuberculosis PtpA
and PtpB) and staph infections (Staphylococcus aureus SaPtpA and SaPtpB).4 Generally, this is
accomplished through interrupting signaling cascades associated with host immune response to
the pathogens. As has been extensively reviewed, despite their implication in a variety of disease
areas, phosphatases have proven to be very difficult targets, as demonstrated by the significant
effort by many pharmaceutical companies to develop inhibitors of PTP1B, an enzyme involved
in insulin signaling.5 Although many PTP1B inhibitors have been reported, very few have made
it to clinical trials, and none of those have been approved for use in the clinic.5a,6
The successful
development of phosphatase inhibitors has three major challenges: first, high potency is difficult
to achieve; second, the highly polar character of pharmacophores required for interaction at the
phosphate binding site can limit both cell permeability and oral bioavailability; and third,
because the catalytic domains of PTPs are highly conserved, achieving inhibitor selectivity can
be challenging. For the development of PTP1B, inhibitors with either sufficient oral availability
or selectivity could be achieved, but attributes could not be attained in tandem.
3
Redox-reversible regulation of phosphatases
One important mechanism of PTP regulation involves the generation of endogenous
hydrogen peroxide (H2O2) by the highly controlled activation of NADPH oxidase enzymes in
response to external stimuli such as growth factors, cytokines and hormones.7,8
Inactivation
occurs first by oxidation of the catalytic cysteine residue to the sulfenic acid (Figure 1.2).9 The
half-life of the sulfenic acid is generally quite low in PTPs, because of its high reactivity, and the
sulfenic acid can often react with a neighboring cysteine to form an intramolecular disulfide (as
with the inhibition of PTEN)10
or with the backbone nitrogen to form a cyclic sulfenamide
species (e.g., PTP1B),11
rendering the PTP inactive. Reaction of the oxidized enzymes with low
molecular weight thiols (e.g., reduced glutathione) or protein thiols (e.g., peroxiredoxin/
thioredoxin cycle) regenerates the active enzyme.12
Figure 1.2. Oxidative inactivation of PTPs. (a) Endogenously produced H2O2 inactivates PTPs by
oxidizing the catalytic cysteine thiolate to the sulfenic acid. The sulfenic acid can then undergo further
reactivity with a neighboring cysteine thiol (b) or a backbone nitrogen (c) to form a disulfide or cyclic
sulfenamide respectively.
Within the last decade, there has been extensive research on the biological and chemical roles
of this cysteine oxidation. The Carroll group, among others, has begun to use the innate
reactivity of the sulfenic acid to develop chemical probes to try to detect and understand the role
of cysteine oxidation in PTPs.13
As these questions begin to be answered it allows for other
possibilities. One aspect that has only just begun to be explored is the possibility of redox active
inhibitors for PTPs. In 2013, Barrios and coworkers reported on a pseudo-irreversible redox
inhibitor of the lymphoid tyrosine phosphatase,14
and in the same year the Thompson group
reported on an inhibitor of a LMW-PTP that utilizes a redox based mechanism.15
Methods for identifying phosphatase inhibitors
High throughput screening
High throughput screening (HTS) refers to the screening of thousands of compounds,
sometimes on the order of hundreds of thousands, in an assay in order to identify lead
4
compounds with nanomolar or low micromolar affinity that can be further optimized to provide
potent inhibitors. This method has been used successfully for many clinically relevant enzyme
targets.16
However, using HTS to target phosphatases has been especially challenging resulting
in a high incidence of false positives. This may be due to a variety of factors, including: (1) the
high reactivity of the active site Cys nucleophile,17
(2) the irreversible oxidation of the catalytic
cysteine,17-18
or (3) the tendency of phosphatase inhibitors, which typically have polar head
groups and hydrophobic tails, to form micelles at high concentrations leading to non-specific
inhibition.19
Researchers at Wyeth have commented on this problem, indicating that in a HTS for
PTP1B inhibition, out of over 6,000 of their initial hits, none were real inhibitors upon cross-
validation.18
X-ray and NMR fragment screens
A few fragment-based approaches have also been applied to phosphatases, namely PTP1B. In
a fragment-based screen, smaller libraries (on the order of thousands of compounds) of low
molecular weight (typically <300 Da) are screened to identify lead compounds to be optimized
into potent lead compounds.20
Because the fragment affinities are in the micromolar to
millimolar ranges, these assays are typically done at higher concentrations than traditional HTS,
which results in a higher incidence of false positives. This has led to the need of more
meticulous, and laborious, methods to directly observe protein binding, rather than enzyme
activity inhibition, for phosphatase targets.
Two rigorous fragment-based methods have been used to achieve potent inhibitors of
PTP1B, NMR and X-ray based fragment screening.21
In the NMR based method, changes in the
protein NMR are detected upon fragment binding.22
Because of the detection of direct binding,
the incidence of false positives can be greatly diminished. In the X-ray based screening method,
fragments are co-crystallized into the active site of the enzyme.20b,c
This method also drastically
decreases the number of false positives in the screen and provides direct binding information.
However, crystal structures take time to acquire and structure activity relationship (SAR)
determination by X-ray methods alone, in particular with enzymes that have no previously
published structural data, can be very slow.23
Even though there has been some success with
NMR and X-ray methods in achieving inhibitors of PTP1B, sufficient oral availability and/or
selectivity was not achieved. Additionally, both methods require a large amount of protein and
dedicated use of expensive instrumentation.
Substrate activity screening
To identify phosphatase inhibitors, the Ellman group has developed an alternative, substrate-
based fragment identification method termed substrate activity screening (SAS).24
This approach
addresses the main limitation in fragment-based screening as it pertains to phosphatases: the
identification of weak binding fragment starting points with high fidelity and efficiency. The
SAS method as it applies to phosphatases is outlined in Scheme 1.1.
In the initial screen, a library of O-aryl phosphate substrates is screened to identify phosphate
substrates. The initial substrate library was synthesized by the Ellman group prior to my arrival
and will not be a subject of discussion in this document, except to say that the library consists of
over 150 low molecular weight (<300 Da), diverse and nonpeptidic O-aryl groups. Once the
5
Scheme 1.1. Substrate activity screening (SAS) method for the identification of phosphatase inhibitors
initial substrate fragments are identified, the O-aryl group can be optimized to achieve more
potent substrates. During optimization the substrates are converted to inhibitors by direct
replacement of the phosphate with known non-hydrolyzable phosphate mimetics.
The key feature of the SAS method when compared to other screening methods is this initial
identification step. In this initial fragment identification, the false positives seen in traditional
HTS methods are eliminated, because active site binding and catalysis are needed for signal
production. Additionally, in traditional HTS methods for inhibitor identification, the desired
output is a decrease in signal, which is typically more difficult to quantify than the increase in
signal observed through the SAS method. Due to catalytic substrate turnover, there is also the
added benefit of signal amplification allowing efficient identification of weak binding substrate
fragments. Finally, because there are many non-hydrolyzable phosphate mimetics (Figure 1.3),25
there is flexibility when it comes time to replace the phosphate moiety with a non-hydrolyzable
replacement.
Figure 1.3. Non-hydrolyzable phosphate mimetics for conversion of substrates to inhibitors.
Enzymatic assays
Substrate screening assay
When using SAS, the initial substrate screen uses an established simple, sensitive and high-
throughput sprectrophotometric-based coupled enzyme assay (Scheme 1.2).26
Because inorganic
phosphate is spectrophotometrically silent, the initial substrate screening assay relies upon the
6
action of a secondary enzyme, purine nucleoside phosphorylase (PNP), which phosphorylates the
ribose ring of nucleoside substrate 1.1, releasing ribose-1-phosphate (1.2) and the UV-active
purine base 1.3. Substrate turnover results in a spectrophotometric shift in maximum absorbance
from 330 nm (substrate 1.1) to 360 nm (product 1.3). Importantly, PNP is selective for inorganic
phosphate and the O-aryl phosphate substrates do not interfere with this enzymatic process.
Therefore, this assay can be used continuously to monitor kinetics of inorganic phosphate
released by the phosphatase-catalyzed hydrolysis of the O-aryl phosphate substrates.
Scheme 1.2. Spectrophotometric coupled assay method for detection of inorganic phosphate released by
PTP of interest
Inhibitor screening assay
Inhibitors are assayed in a standard inhibition assay for phosphatases using p-nitrophenyl
phosphate (pNPP) as a chromogenic substrate (Scheme 1.3).27
This is a competitive inhibition
assay in which the absorbance of 1.4 is continuously monitored at 405 nm as pNPP is hydrolyzed
by the phosphatase of interest. The concentration of pNPP is held constant, while the
concentration of the inhibitor is varied, resulting in a dose-response curve from which Ki values
can be determined. Importantly, assays are performed with the addition of Triton X-100
detergent (0.004% to 0.01% total volume) to prevent non-specific aggregation-based inhibition.19
7
Scheme 1.3. Spectrophotometric assay method for PTP inhibitors
References
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9
Chapter 2. Identification of inhibitors of the Mycobacterium tuberculosis phosphatase PtpA
Abstract: This chapter focuses on the identification of phosphatase inhibitors of Mycobacterium
tuberculosis protein tyrosine phosphatase PtpA. Inhibitors incorporating the well established
non-hydrolyzable phosphate mimic, the difluoromethylenephosphonic acid (DFMP), were
explored, resulting in low micromolar inhibitors of PtpA. The basic scaffold of the inhibitors was
identified through the use of the SAS fragment based method by assaying an O-aryl substrate
phosphate library that had previously been generated within the Ellman group. The most potent
inhibitor was also shown to be selective for PtpA over a variety of human phosphatases as well
as Mycobacterium tuberculosis protein tyrosine phosphatase PtpB. This inhibitor represents a
chemical tool that can be used in conjunction with PtpB selective inhibitors described previously
within the Ellman group to further probe the roles of PtpA and PtpB in tuberculosis infection.
The majority of this work has been published (Rawls, K. A.; Lang, P. T.; Takeuchi, J.; Imamura,
S.; Baguley, T. D.; Grundner, C.; Alber, T.; Ellman, J. A., Bioorg. Med. Chem. Lett. 2009, 19,
6851).
10
Authorship
This work was conducted in collaboration with Dr. Katherine Rawls, Dr. Jun Takeuchi, Dr.
Shinichi Imamura, Dr. P. Therese Lang, and Dr. Christoph Grundner. The inhibitor library was
made by Dr. Katherine Rawls, Dr. Jun Takeuchi, Dr. Shinichi Imamura and myself. Dr.
Grundner provided enzyme for the assays, Dr. Lang performed all modeling studies, and Dr.
Rawls and I performed all substrate and inhibitor assays.
Introduction
Tuberculosis (TB) is a chronic, infectious disease caused by Mycobacterium tuberculosis
(Mtb), and is second only to HIV/AIDS as the greatest killer worldwide due to a single infectious
agent. In 2012, 8.6 million people contracted TB, and the disease was responsible for 1.3 million
deaths.1 The current treatment of drug-sensitive strains requires 6–9 months to fully eradicate the
infection. Current frontline treatments2 are hindered by the requirement to penetrate the
unusually thick mycobacterial cell wall3 in order to be effective, and only one drug has been
approved for treatment of TB since 1960.1 Compounding the problem is the development of
drug-resistant strains, caused in large part by a lack of compliance with the lengthy treatment
regimen.1 New Mtb drugs that act on novel targets are needed to shorten treatment time and
address the emergence of antibiotic resistance.
Ever since the discovery of the Yersinia PTP YopH, many host-pathogen interactions have
been found to be dependent on pathogen-secreted phosphatases.4 Mtb encodes three secreted
phosphatases, two of which are protein tyrosine phosphatases, PtpA and PtpB, that are promising
new targets for TB drug development.5 Although genetic deletion of ptpA or ptpB does not affect
Mtb growth in culture,6 it has been shown that these secreted phosphatases not only shut down
critical host cellular processes, but also promote Mtb survival within host macrophages.7 In
particular, PtpA inhibits phagosome acidification and maturation by blocking the recruitment of
the vacuolar H+-ATPase.
7a-b Although not a classical drug target because it is not essential in
vitro, targeting the secreted PtpA in the host macrophage circumvents two central resistance
mechanisms of Mtb; i.e. poor drug permeability due to the Mtb cell wall,3 and pump-mediated
drug efflux.8
Targeting PtpA is an attractive strategy which, in theory, would reduce the ability of Mtb
bacteria to grow and survive in the infected host, thus reducing the lengthy treatment time. Very
few PtpA inhibitors have been reported in the literature thus far, and the novel inhibitors
described in this chapter represent some of the most potent and selective compounds reported to
date.5
Initial scaffold identification
Identification of inhibitors for PtpA began with screening of a previously synthesized 150-
member O-aryl phosphate library.9 On first inspection, it was apparent that ortho and meta
substitution on the substrate fragments was not tolerated as exemplified by substrates 2.01 and
2.02 (Figure 2.1). Importantly, there were several substrate fragment hits with higher affinity
than the simple phenyl phosphate 2.03. Due to its improved solubility over compound 2.07 and it
amenability to modification over 2.08, compound 2.06 was chosen as the initial hit for further
compound optimization.
11
Figure 2.1. Selected results from the initial O-aryl phosphate substrate screening against PtpA.
Conversion to inhibitors
Substrate scaffold 2.06 was converted into inhibitor analogs 2.09 and 2.1010
(Figure 2.2), but
only the non-hydrolyzable difluoromethylenephosphonic acid (DFMP) isostere11
(2.09) was
active against PtpA. Despite the dianionic character of the DFMP isostere, it has been shown to
be both cell permeable and orally bioavailable in animals.12
Compounds containing this isostere
can be easily prepared from advanced synthetic intermediates allowing for rapid analog synthesis
using modified literature procedures.13
Figure 2.2. Conversion of scaffold 2.06 to inhibitors using DFMP and isoxazolecarboxylic acid isosteres
and inhibitory activity against PtpA.
Inhibitor library synthesis and evaluation
Synthesis of benzamide DFMP inhibitors began with a three step sequence to afford
advanced intermediate acid 2.14 (Scheme 2.1).14
Benzyl protection of the commercially available
4-iodobenzoic acid 2.11, followed by a copper mediated organozinc addition with the
commercial diethyl(bromodifluoromethyl)phosphonate affords compound 2.13. Hydrogenolysis
of the benzyl group restores the acid functionality to achieve key intermediate 2.14. With the
acid in hand, a number of commercial amines containing a variety of functionality were
incorporated through amide bond formation, followed by deprotection of the diethyl phosphonate
to afford the desired inhibitors (2.16).
12
Scheme 2.1. Synthesis of DFMP benzamide inhibitors
Upon assaying the initial library of DFMP amide inhibitors against PtpA (Table 2.1),
benzanilide 2.09, the initial inhibitor derived from substrate 2.06 (Figure 2.1), was identified as
the most potent compound (Ki = 24.0 M). Alkyl substituted benzamide compounds 2.17–2.21
showed minimal activity in the assay. The only alkyl derivative that even showed modest activity
was compound 2.23 (Ki = 49.7 M), presumably due to the increased acidity of the NH group
when compared to the other alkyl analogs. Acid analog 2.25 (Figure 2.3) also showed poor
activity, indicating the need of an amide and/or aromatic functionality for activity. However, the
placement of the aromatic group also seems to be important as the N-benzyl substituted 2.24 (Ki
= 43.1 M) was half as potent as 2.09, and the N-phenethyl substituted 2.22, was even less
potent (Ki = 61.6 M).
Amide replacement analog synthesis and evaluation
To investigate the importance of the amide moiety and positioning, we synthesized several
amide replacement analogs (Scheme 2.2). We thought that the position of the amide as well as
the availability of a hydrogen bond donor and acceptor from the amide may play an important
role in binding affinity. We therefore thought to examine the inversion of the amide direction
(2.29), addition of a methylene linker (2.33), removal of the carbonyl (2.36), replacement of
nitrogen with other heteroatoms (2.39), methylation of the amide nitrogen (2.41) and the
replacement of the carbonyl with a sulfonamide group (2.45). Each analog was synthesized in a
fashion similar to the DFMP benzamide inhibitors, but with varying the coupling partners at each
stage of the synthesis. Reverse amide compound 2.29 was synthesized by starting with
commercially available 1-iodo-4-nitrobenzene 2.26 and coupling it with
diethyl(bromodifluoromethyl)phosphonate, followed by hydrogenation to afford amine 2.28,
amide coupling with benzoyl chloride and deprotection to arrive at 2.29. The extended
homoamide 2.33 could be achieved by coupling the commercially available acid chloride 2.30
with aniline followed by the same coupling and deprotection sequence. Amine analog 2.36 was
13
Table 2.1. Initial DFMP amide inhibitor screens against PtpAa
compound R Ki (M) compound R Ki (M)
2.09
24.0 2.21
72.4
2.17 >100 2.23 49.7
2.18
>100 2.24
43.1
2.19
>100 2.22
61.6
2.20
>100
aKi values were determined using at least two independent measurements.
Figure 2.3. Free acid containing DFMP inhibitor screening result against PtpA.
achieved through a reductive amination between 4-iodobenzaldehyde and aniline, followed by
the common coupling and deprotection sequence. Ether 2.39 was synthesized first with a
Mitsunobu reaction between 4-iodobenzyl alcohol and phenol, followed by the same coupling
and deprotection. The N-methylated derivative is synthesized following the previously described
amide coupling of acid 2.14 and N-methylaniline, followed by deprotection of the
diethylphosphonate. Finally, the sulfonamide derivative was synthesized by coupling
commercially available 4-iodobenzenesulfonyl chloride 2.42 with aniline, which is likewise
followed by the coupling and deprotection sequence.
Scheme 2.2. Synthesis of amide replacement analogs 2.29, 2.33, 2.36, 2.39, 2.41 and 2.45
14
15
Each of the amide analogs was tested for activity versus PtpA (Table 2.2), and it was found
that all amide replacements resulted in loss of activity. This could be due to a loss of hydrogen
bonding interactions (2.36, 2.39, 2.41), or to changes associated with the entropic energies of
binding due to an increase in the number of rotatable bonds (2.33, 2.36, 2.39). Interestingly, the
only analog in this series to show any activity was the reverse amide 2.29. It is expected that the
aryl ring would be similarly positioned and only the hydrogen bonding characteristics of the
molecule would be perturbed. These results confirmed that the benzanilide scaffold 2.09 was the
best scaffold to optimize in our further efforts.
Table 2.2. Evaluation of amide replacement analogs for PtpA inhibition
a
compound structure Ki (M) compound Structure Ki (M)
2.29
65.5 2.39
>100
2.33
>100 2.41
>100
2.36
>100 2.45
>100
aKi values were determined using at least two independent measurements.
Benzanilide scaffold optimization and evaluation
Because benzanilide 2.09 was identified as the most potent analog in the inhibition screens
against PtpA, and the amide moiety was shown to be important for potency, a focused
benzanilide library was synthesized in order to determine an SAR and to improve potency of the
compounds (Table 2.3). All focused library members were synthesized in the route discussed for
the benzamide library (Scheme 2.1. Synthesis of DFMP benzamide inhibitors) using
commercially available and diversely substituted anilines. Electron withdrawing groups
generally provided more potent compounds than compounds containing electron donating or
16
neutral substituents (data not shown). Additionally, substitution at the meta and para positions
(2.50–2.51, 2.52–2.55, 2.57–2.58) resulted in compounds with higher potency than substitution
at the ortho position (2.46–2.49). Combining the favorable substituents resulted in compounds
2.56 and 2.59–2.63 which all were more potent than the lead benzanilide 2.09 (24.0 ± 0.9 M).
The most potent of these compounds, 2.63, had a Ki of 1.4 ± 0.3 M.
Table 2.3. Focused benzanilide library screen against PtpA
a
cpd R Ki (M) cpd R Ki (M) cpd R Ki (M)
2.09
24.0 ± 0.9 2.52
22.7 ± 3.2 2.59
6.7 ± 0.4
2.46
>100 2.53
18.5 ± 2.5 2.60
6.0 ± 0.4
2.47
68.0 ± 6.0 2.54
18.2 ± 0.9 2.61
4.9 ± 1.7
2.48
54.8 ± 14.5 2.55
16.8 ± 7.0 2.62
3.3 ± 0.6
2.49
44.6 ± 7.4 2.56
11.4 ± 0.3 2.63
1.4 ± 0.3
2.50
41.8 ± 5.8 2.57
10.7 ± 1.2
2.51
34.9 ± 3.5 2.58
10.3 ± 1.0
aKi values were determined using at least two independent measurements.
In order to rule out non-specific aggregation-based inhibition,15
compound 2.63 was tested
with two different concentrations of enzyme (300 and 600 nM) and two different concentrations
of the detergent Triton X-100 (0.004% and 0.01%, Figure 2.4). The Ki values remained constant
(within experimental error) for each of these conditions, consistent with active site competitive
inhibition. Additionally, the inhibition curves were found to have a Hill coefficient of h = –1.0 ±
0.1, also indicating that the inhibitor was binding into a single enzyme site rather than non-
specific aggregation-based inhibition.
17
Figure 2.4. Ki values of inhibitor 2.63 at two concentrations of PtpA and two concentrations of Triton X-
100 detergent. Ki values were found to be independent of either parameter, indicating real, and not
aggregation-based, inhibition.
Inhibitor selectivity profile
Achieving inhibitor selectivity is a major challenge because of the high structural homology
between PTP active sites.16
Gratifyingly, compound 2.63 was found to be highly selective (>70-
fold) when tested against a panel of PTPs and DUSPs, including TC-Ptp, an essential human
phosphatase important for immune response17
(Table 2.4). This was not unexpected, as low
molecular weight PTPs share little structural homology with classical PTPs and DUSPs.18
However, compound 2.63 was also found to be 11-fold selective over the human low molecular
weight phosphatase, HCPtpA, which shows 38% sequence identity to Mtb PtpA.19
Finally,
compound 2.63 did not inhibit Mtb PtpB; this selectivity should enable the use of this inhibitor as
a tool compound in order to further study the biochemical role of PtpA.
Table 2.4. Selectivity profile of inhibitor 2.63 against a panel of human and Mtb PTPs
a
Mtb PTPs Human PTPs
PtpA PtpB PTP1B TC-Ptp VHR CD45 LAR HCPtpA
Ki (M) 1.4 ± 0.3 >100 >100 >100 >100 >100 >100 14.8 ± 1.9
selectivity -- >70 >70 >70 >70 >70 >70 11 aKi values were determined using at least two independent measurements.
18
Modeling studies
Finally, using AMBER 920
and DOCK 6.4,21
a molecular model of all PtpA inhibitors bound
in the active site of a previously published apo crystal structure of PtpA (PDB ID: 1U2P)19
was
generated (Figure 2.5). The published crystal structure of apo-PtpA was first relaxed using
molecular dynamics in AMBER, followed by docking the compounds into the PtpA active site
with DOCK 6.4. Each of the compounds, including the initial lead benzanilide 2.09 and
optimized inhibitor 2.63, docked such that the DFMP warhead was in direct contact with the
catalytic residues of the protein. Additionally, the scoring function of the docking program
ranked the compounds in the same general order observed experimentally (data not shown),
indicating that the model is reasonably accurate.
Each of the docked compounds exhibited significant hydrogen bonding interactions with
PtpA (Figure 2.5). Nine hydrogen bonds were found between compound 2.09 and PtpA active
site residues, versus seven for compound 2.63. This model of inhibitor-enzyme interactions also
predicted varying degrees of pi-stacking with Trp48, the effectiveness of which depended on the
orientation of the aryl ring and its resulting ability to overlap with the indole ring of Trp48
(Figure 2.5a-b). This binding mode was not completely unexpected, since Trp pi-stacking has
been previously observed in inhibitor-enzyme complexes.22
Further development of SAR around
the benzanilide scaffold could lead to compounds with improved affinity compared to
benzanilide 2.63. In particular, modifications to further improve potency could include
improving pi-stacking efficiency with Trp48, as well as introduction of functionality off of the
pendant anilide ring to extend into an adjacent unfilled enzyme pocket (Figure 2.5c).
Figure 2.5. Model of (a) parent benzanilide 2.09 and (b) optimized benzanilide 2.63 docked in the active
site of PtpA (PDB ID: 1U2P)19
using DOCK 6.4.21
Hydrogen bonds (green lines) between each inhibitor
and active site residues are shown. Trp48 is emphasized to show pi-stacking interactions with each
inhibitor. Also shown is the PtpA binding pocket with inhibitor 2.63-enzyme contact points shown in blue
(c). The arrow indicates the position of an unfilled enzyme pocket adjacent to the docked inhibitor.23
Conclusions
In conclusion, we have identified inhibitors with single-digit micromolar affinity for PtpA
based on the benzanilide scaffold 2.09. SAR optimization resulted in compound 2.63, which,
nearly five years after publication of this work, still represents the most potent and selective
PtpA inhibitor in the literature to date.5
Compound 2.63 was found to be over 70-fold selective
for PtpA over a panel of human PTPs and DUSPs, and 11-fold selective over the highly
19
homologous human HCPtpA, which shows 38% sequence identity to Mtb PtpA. Molecular
modeling highlighted the importance of pi-stacking with Trp48, and hydrogen bonding with
active-site residues. Finally, 2.63, was found to be selective over Mtb PtpB, which allows it to be
a valuable tool compound, along with the PtpB selective inhibitor previously reported by our
group,9 in order to probe the biochemical roles of the Mtb PTPs.
Experimental
General synthetic methods
Unless otherwise noted, all reagents were obtained from commercial suppliers and used
without further purification. Tetrahydrofuran (THF), dichloromethane (CH2Cl2), toluene, and
diethyl ether (Et2O) were dried over alumina under a nitrogen atmosphere. Solvents used for
reactions set up in a nitrogen-filled inert atmosphere box, including THF and toluene, were
additionally degassed with three consecutive freeze pump thaw cycles and stored over 3Å
molecular sieves. Methanol was dried over calcium hydride under a nitrogen atmosphere. All
reactions, unless otherwise stated, were performed under inert atmosphere using syringe,
cannula, and Schlenk techniques, or set up in a nitrogen-filled inert atmosphere box, with flame
or oven-dried glassware. All 1H,
13C,
19F, and
31P NMR spectra were measured with a Bruker
DRX-500, AVB-400, AVQ-400 or AV-300 spectrometer. NMR chemical shifts are reported in
ppm relative to 1,2-difluorobenzene (–138.9) for 19
F NMR and trimethylphosphate (3.0) for 31
P
NMR. Mass spectrometry (HRMS) was carried out by the University of California, Berkeley
Mass Spectrometry Facility.
Synthesis and analytical data for DFMP inhibitors
Synthesis and analytical data for benzamide inhibitor 2.63
Compound 2.13. Compound 2.13 was synthesized according to modified literature
procedures.14
A solution of diethyl(bromodifluoromethyl)phosphonate (13.80 g, 44.0 mmol) in
DMA (20 mL) in a flame-dried 50 mL flask under an N2 atmosphere was slowly added to a
stirred suspension of activated Zn dust (2.88 g, 44.0 mmol) in DMA (20 mL) in a flame-dried
250 mL flask under an N2 atmosphere at 60 °C via cannula addition. After addition was
complete, the resulting mixture was sonicated at ambient temperature for 3 h, followed by
addition of CuBr (6.31 g, 44.0 mmol) in one portion. A solution of benzyl 4-iodobenzoate 2.1214
(5.41 g, 16.0 mmol) in DMA (5 mL) was added dropwise, and the resulting mixture was stirred
for 38 h at ambient temperature. The mixture was diluted with water (50 mL) and Et2O (50 mL),
and was then passed through Celite. The organic layer was separated, washed with brine (1 x 100
mL), dried over anhydrous MgSO4, and filtered. The solvent was removed under reduced
pressure to afford crude product, which was then purified via column chromatography to yield
20
2.13 as a colorless oil (4.68 g, 73% yield). 1H NMR (400 MHz, CDCl3): δ 8.16 (d, J = 8.0 Hz,
2H), 7.70 (d, J = 8.0 Hz, 2H), 7.46–7.36 (m, 5H), 5.39 (s, 2H), 4.26–4.11 (m, 4H), 1.30 (t, J = 7.2 Hz, 6H);
19F NMR (376 MHz, CDCl3): δ –108.59 (d, JPF = 109 Hz);
31P NMR (162 MHz,
CDCl3): δ 5.76 (t, JPF = 109 Hz). HRMS-FAB (m/z): [M+H]+ calcd. for C19H22F2O5P, 399.1172;
found, 399.1177.
Compound 2.14. Compound 2.14 was synthesized by modified literature procedures.
14 A
solution of 2.13 (2.50 g, 6.28 mmol) in MeOH (5 mL) was added to 10% Pd/C (835 mg, ca. 50%
wet) in MeOH (30 mL). The reaction mixture was stirred for 16 h under H2 atmosphere. The
catalyst was then removed by filtration through Celite, and the solvent was removed under
reduced pressure to give crude 2.14. The crude product was purified by recrystallization from
EtOAc/hexanes to yield 2.14 as a white powder (3.67 g, 86% yield). 1H NMR (400 MHz,
CD3OD): δ 8.15 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 8.0 Hz, 2H), 4.26–4.13 (m, 4H), 1.31 (t, J = 7.2
Hz, 6H); 19
F NMR (376 MHz, CD3OD): δ –111.51 (d, JPF = 109 Hz); 31
P NMR (162 MHz,
CD3OD): δ 5.73 (t, JPF = 109 Hz). HRMS-FAB (m/z): [M+Na]+ calcd. for C12H15F2O5PNa,
331.0523; found, 331.0531.
Compound 2.64. To a solution of 2.14 (98 mg, 0.32 mmol) in dry CH2Cl2 (3 mL) in a flame-
dried 10 mL flask under an N2 atmosphere was added oxalyl chloride (55 L, 0.64 mmol) and a
catalytic amount of DMF. The reaction mixture was stirred for 1 h at ambient temperature,
followed by removal of the solvent under reduced pressure, and drying under high vacuum. The
resulting acid chloride was dissolved in dry CH2Cl2 (2 mL) under an N2 atmosphere and slowly
added to a solution of 4-bromo-3,5-bis(trifluoromethyl)aniline (114 mg, 0.37 mmol) and
triethylamine (67 L, 0.48 mmol) in dry CH2Cl2 (3 mL) in a flame-dried 25 mL flask under an
N2 atmosphere. The reaction mixture was stirred at ambient temperature for 18 h. The solvent
was evaporated under reduced pressure to afford crude 2.64. The crude product was purified via
column chromatography to give 2.64 as a white solid (96 mg, 50% yield). 1H NMR (400 MHz,
CD3OD): δ 9.07 (br s, 1H), 8.40 (s, 2H), 7.92 (d, J = 8.2 Hz, 2H), 7.65 (d, J = 7.8 Hz, 2H), 4.29–
4.16 (m, 4H), 1.33 (t, J = 7.1 Hz, 6H); 19
F NMR (376 MHz, CD3OD): δ –63.47, –108.27 (d, JPF
= 114 Hz); 31
P NMR (162 MHz, CD3OD): δ 4.86 (t, JPF = 114 Hz). MS-ESI (m/z): [M+H]+
calcd. for C20H18BrF8NO4P, 596.99; found, 598.0 and 599.0.
21
Inhibitor 2.63. To a stirred solution of 2.64 (35 mg, 0.06 mmol) in CHCl3 (3 mL) was added
TMSI (33 L, 0.22 mmol). The mixture was stirred for 3 h at ambient temperature. Volatiles
were removed under reduced pressure and the residue was dissolved in MeOH (3 mL) and stirred
at ambient temperature for 18 h. The solvent was removed under reduced pressure to give crude
2.63. The crude product was purified by recrystallization from EtOAc/hexanes to yield 2.63 as a white powder (13 mg, 41% yield).
1H NMR (400 MHz, CD3OD): δ 8.54 (s, 2H), 8.06 (d, J = 8.0
Hz, 2H), 7.77 (d, J = 8.0 Hz, 2H); 19
F NMR (376 MHz, CD3OD): δ –64.40, –112.46 (d, JPF =
109 Hz); 31
P NMR (162 MHz, CD3OD): δ 2.55 (t, JPF = 109 Hz). HRMS-FAB (m/z): [M+H]+
calcd. for C16H10[79]
BrF8NO4P, 541.9403; found, 541.9406.
General synthesis of other benzamide inhibitors
Benzamide inhibitors 2.09, 2.17–2.24, 2.46–2.62 and 2.41 were synthesized by following the
general procedures described for the synthesis of 2.63, using commercially available amines or
anilines.
Analytical data for benzamide inhibitors
Inhibitor 2.09.
1H NMR (400 MHz, CD3OD): δ 8.02 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 8.0 Hz,
2H), 7.69 (d, J = 8.0 Hz, 2H), 7.36 (t, J = 7.6 Hz, 2H), 7.15 (t, J = 7.6 Hz, 1H); 19
F NMR (376 MHz, CD3OD): δ –112.39 (d, JPF = 109 Hz);
31P NMR (162 MHz, CD3OD): δ 2.65 (t, JPF = 109
Hz). HRMS-FAB (m/z): [M+Na]+ calcd. for C14H12F2NO4PNa, 350.0370; found, 350.0366.
Inhibitor 2.17.
1H NMR (400 MHz, CD3OD): δ 7.89 (d, 2H, J = 8.1 Hz), 7.68 (d, 2H, J = 8.1
Hz), 3.09 (s, 3H); 19
F NMR (376 MHz, CD3OD): δ –110.67 (d, JPF = 110 Hz); 31
P NMR (162
MHz, CD3OD): δ 4.34 (br t, JPF = 110 Hz). HRMS-ESI (m/z): [M+H]+ calcd. for C9H11O4NF2P,
266.0388; found, 266.0385.
22
Inhibitor 2.18.
1H NMR (400 MHz, CD3OD): δ 7.89 (d, 2H, J = 8.3 Hz), 7.69 (d, 2H, J = 8.3
Hz), 3.20 (d, 2H, J = 6.9 Hz), 1.93 (nonet, 1H, J = 6.9 Hz), 0.96 (d, 6H, J = 6.9 Hz); 19
F NMR (376 MHz, CD3OD): δ –110.60 (d, JPF = 110 Hz);
31P NMR (162 MHz, CD3OD): δ 4.37 (br t,
JPF = 112 Hz). HRMS-ESI (m/z): [M+H]+ calcd. for C12H17O4NF2P, 308.0858; found, 308.0852.
Inhibitor 2.19.
1H NMR (400 MHz, CD3OD): δ 7.89 (d, 2H, J = 7.1 Hz), 7.69 (d, 2H, J = 7.1
Hz), 3.22 (d, 2H, J = 6.9 Hz), 3.77 (br m, 4H), 3.71–3.62 (br m, 2H), 1.34–1.18 (br m, 3H), 1.02 (br m, 2H);
19F NMR (376 MHz, CD3OD): δ –110.65 (d, JPF = 109 Hz);
31P NMR (162 MHz,
CD3OD): δ 4.63 (br m). HRMS-ESI (m/z): [M+H]+ calcd. for C15H21O4NF2P, 348.1171; found,
348.1166.
Inhibitor 2.20.
1H NMR (400 MHz, CD3OD): δ 7.85 (d, 2H, J = 8.3 Hz), 7.70 (d, 2H, J = 8.3
Hz), 4.13 (m, 1H), 4.03 (quintet, 2H, J = 7.1 Hz), 3.43 (br m, 2H), 3.12 (br m, 2H), 2.17 (br m, 2H), 3.79 (br m, 2H), 1.24 (t, 3H, J = 7.1 Hz);
19F NMR (376 MHz, CD3OD): δ –108.82 (d, JPF =
99 Hz); 31
P NMR (162 MHz, CD3OD): δ 3.20 (br t, JPF = 99 Hz). HRMS-ESI (m/z): [M+H]+
calcd. for C15H22O4N2F2P, 363.1280; found, 363.1273.
Inhibitor 2.21.
1H NMR (400 MHz, CD3OD): δ 7.87 (d, 2H, J = 8.0 Hz), 7.67 (d, 2H, J = 8.0
Hz), 3.88–3.84 (m, 1H), 1.95 (br m, 2H), 3.81 (br m, 2H), 3.68 (br m, 1H), 3.45–1.29 (br m, 4H),
1.27–1.19 (br m, 1H); 19
F NMR (376 MHz, CD3OD): δ –110.63 (d, JPF = 110 Hz); 31
P NMR
(162 MHz, CD3OD): δ 4.72 (br m). HRMS-ESI (m/z): [M+H]+ calcd. for C14H19O4NF2P,
334.1014; found, 334.1020.
23
Inhibitor 2.22.
1H NMR (400 MHz, CD3OD): δ 7.94 (d, 2H, J = 8.2 Hz), 7.84 (d, 2H, J = 8.2
Hz), 7.34–7.17 (m, 5H), 3.60 (t, 2H, J = 7.4 Hz), 2.91 (t, 2H, J = 7.4 Hz); 19
F NMR (376 MHz, CD3OD): δ –110.73 (d, JPF = 112 Hz);
31P NMR (162 MHz, CD3OD): δ 4.49 (br m). HRMS-ESI
(m/z): [M+H]+ calcd. for C16H17O4NF2P, 356.0858; found, 356.0865.
Inhibitor 2.23.
1H NMR (400 MHz, CD3OD): δ 7.93 (d, 2H, J = 7.6 Hz), 7.72 (d, 2H, J = 8.0
Hz), 4.10 (q, 2H, JHF = 9.2 Hz); 19
F NMR (376 MHz, CD3OD): δ –72.92 (t, JFH = 9.4 Hz),
–110.78 (d, JPF = 109 Hz); 31
P NMR (162 MHz, CD3OD): δ 4.48 (br m). HRMS-ESI (m/z): [M–
H]– calcd. for C10H8O4NF5P, 332.0117; found, 332.0105.
Inhibitor 2.24.
1H NMR (400 MHz, CD3OD): δ 7.93 (d, 2H, J = 8.1 Hz), 7.70 (d, 2H, J = 8.1
Hz), 7.36–7.22 (m, 5H), 4.58 (s, 2H); 19
F NMR (376 MHz, CD3OD): δ –110.57 (d, JPF = 109
Hz); 31
P NMR (162 MHz, CD3OD): δ 4.09 (br t, JPF = 109 Hz). HRMS-ESI (m/z): [M+H]+ calcd.
for C15H15O4NF2P, 342.0701; found, 342.0712.
Inhibitor 2.46.
1H NMR (400 MHz, CD3OD): δ 8.03 (d, J = 7.1 Hz, 2H), 7.80–7.71 (m, 3H),
7.29–7.14 (m, 3H); 19
F NMR (376 MHz, CD3OD): δ –110.70 (d, JPF = 111 Hz), –124.20; 31
P
NMR (162 MHz, CD3OD): δ 4.27 (t, JPF = 102 Hz). MS-ESI (m/z): [2M+H]+ calcd. for
C28H23F6N2O8P2, 691.08; found, 691.0.
24
Inhibitor 2.47.
1H NMR (400 MHz, CD3OD): δ 8.02 (d, J = 8.0 Hz, 2H), 7.89 (t, J = 2.0 Hz,
1H), 7.75 (d, J = 8.0 Hz, 2H), 7.60 (dd, J = 8.0, 2.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.15 (dd, J = 8.0, 2.0 Hz, 1H);
19F NMR (376 MHz, CD3OD): δ –112.44 (d, JPF = 109 Hz);
31P NMR (162
MHz, CD3OD): δ 2.64 (t, JPF = 109 Hz). HRMS-EI (m/z): [M+H]+ calcd. for
C14H12[35]
ClF2NO4P, 362.0160; found, 362.0169.
Inhibitor 2.48.
1H NMR (400 MHz, CD3OD): δ 8.03 (d, J = 8.0 Hz, 2H), 7.78–7.71 (m, 4H),
7.62–7.60 (m, 1H), 7.54–7.52 (m, 1H); 19
F NMR (376 MHz, CD3OD): δ –61.52, –110.88 (d, JPF
= 109 Hz); 31
P NMR (162 MHz, CD3OD): δ 2.60 (t, JPF = 109 Hz). HRMS-FAB (m/z): [M+H]+
calcd. for C15H12F5NO4P, 396.0424; found, 396.0424.
Inhibitor 2.49.
1H NMR (400 MHz, CD3OD): δ 8.07 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 8.0 Hz,
2H), 7.72–7.69 (m, 2H), 7.43 (t, J = 7.6 Hz, 1H), 7.21 (d, J = 7.6 Hz, 1H); 19
F NMR (376 MHz, CD3OD): δ –112.45 (d, JPF = 109 Hz);
31P NMR (162 MHz, CD3OD): δ 2.64 (t, JPF = 109 Hz).
HRMS-FAB (m/z): [M+H]+ calcd. for C14H12
[79]BrF2NO4P, 405.9655; found, 405.9666.
Inhibitor 2.50.
1H NMR (400 MHz, CD3OD): δ 8.02 (d, J = 8.0 Hz, 2H), 7.75 (d, J = 8.0 Hz,
2H), 7.69–7.66 (m, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.38–7.32 (m, 1H), 6.88 (t, J = 8.0 Hz, 1H); 19
F NMR (376 MHz, CD3OD): δ –112.41 (d, JPF = 109 Hz), –115.00; 31
P NMR (162 MHz,
CD3OD): δ 2.80 (t, JPF = 109 Hz). HRMS-FAB (m/z): [M+H]+ calcd. for C14H10F3NO4P,
344.0300; found, 344.0292.
25
Inhibitor 2.51.
1H NMR (400 MHz, CD3OD): δ 8.02 (d, J = 8.0 Hz, 2H), 7.89 (t, J = 2.0 Hz,
1H), 7.75 (d, J = 8.0 Hz, 2H), 7.60 (dd, J = 8.0, 2.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.15 (dd, J = 8.0, 2.0 Hz, 1H);
19F NMR (376 MHz, CD3OD): δ –112.44 (d, JPF = 109 Hz);
31P NMR (162
MHz, CD3OD): δ 2.64 (t, JPF = 109 Hz). HRMS-EI (m/z): [M+H]+ calcd. for
C14H12[35]
ClF2NO4P, 362.0160; found, 362.0169.
Inhibitor 2.52.
1H NMR (400 MHz, CD3OD): δ 8.01 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 8.0 Hz,
2H), 7.70 (dd, J = 8.8, 4.8 Hz, 2H), 7.10 (t, J = 8.8 Hz, 2H); 19
F NMR (376 MHz, CD3OD): δ
–112.38 (d, JPF = 109 Hz), –120.72; 31
P NMR (162 MHz, CD3OD): δ 2.71 (t, JPF = 109 Hz).
HRMS-FAB (m/z): [M+Na]+ calcd. for C14H10F3NO4PNa, 390.0095; found, 390.0102.
Inhibitor 2.53.
1H NMR (400 MHz, CD3OD): δ 8.03 (d, J = 8.0 Hz, 2H), 7.94 (d, J = 8.0 Hz,
2H), 7.76 (d, J = 8.0 Hz, 2H), 7.66 (d, J = 8.0 Hz, 2H); 19
F NMR (376 MHz, CD3OD): δ –64.41,
–112.37 (d, JPF = 109 Hz); 31
P NMR (162 MHz, CD3OD): δ 2.59 (t, JPF = 109 Hz). HRMS-FAB
(m/z): [M+H]+ calcd. for C15H12F5NO4P, 396.0424; found, 396.0423.
Inhibitor 2.54.
1H NMR (400 MHz, CD3OD): δ 8.03–8.00 (m, 4H), 7.75 (d, J = 8.0 Hz, 1H),
7.67–7.65 (m, 1H), 7.29–7.26 (m, 2H); 19
F NMR (376 MHz, CD3OD): δ –112.27 (d, JPF = 109
Hz); 31
P NMR (162 MHz, CD3OD): δ 2.64 (t, JPF = 109 Hz). HRMS-FAB (m/z): [M+H]+
calcd.
for C14H12[79]
BrF2NO4P, 405.9655; found, 405.9650.
26
Inhibitor 2.55.
1H NMR (400 MHz, CD3OD): δ 8.01 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 8.0 Hz,
2H), 7.72 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H); 19
F NMR (376 MHz, CD3OD): δ –112.42
(d, JPF = 109 Hz); 31
P NMR (162 MHz, CD3OD): δ 2.60 (t, JPF = 109 Hz). HRMS-ESI (m/z):
[M+2Na–H]+ calcd. for C14H10
[35]ClF2NO4PNa2, 405.9799; found, 405.9815.
Inhibitor 2.56.
1H NMR (400 MHz, CD3OD): δ 8.14 (m, 1H), 8.05–7.92 (m, 3H), 7.82 (s,
1H), 7.75 (m, 1H), 7.31 (m, 1H). MS-ESI (m/z): [M–H]– calcd. for C15H9F6NO4P, 413.0252;
found 413.0.
Inhibitor 2.57.
1H NMR (400 MHz, CD3OD): δ 8.17 (s, 1H), 8.04 (d, J = 8.0 Hz, 2H), 7.95
(d, J = 8.0 Hz, 1H), 7.76 (d, J = 8.0 Hz, 2H), 7.56 (t, J = 8.0 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H); 19
F NMR (376 MHz, CD3OD): δ –65.08, –112.39 (d, JPF = 109 Hz); 31
P NMR (162 MHz,
CD3OD): δ 2.64 (t, JPF = 109 Hz). HRMS-FAB (m/z): [M+Na]+ calcd. for C15H11F5NO4PNa,
418.0244; found, 418.0243.
Inhibitor 2.58.
1H NMR (400 MHz, CD3OD): δ 8.01 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 8.0 Hz,
2H), 7.67 (d, J = 8.8 Hz, 2H), 7.50 (d, J = 8.8 Hz, 2H); 19
F NMR (376 MHz, CD3OD): δ –112.39
(d, JPF = 109 Hz); 31
P NMR (162 MHz, CD3OD): δ 2.74 (t, JPF = 109 Hz). HRMS-FAB (m/z):
[M+H]+ calcd. for C14H12
[79]BrF2NO4P, 405.9655; found, 405.9650.
27
Inhibitor 2.59.
1H NMR (400 MHz, CD3OD): δ 8.06 (d, J = 2.4 Hz, 1H), 8.01 (d, J = 8.0 Hz,
2H), 7.75 (d, J = 8.0 Hz, 2H), 7.64 (dd, J = 8.8, 2.4 Hz, 1H), 7.50 (d, J = 8.8 Hz, 1H); 19
F NMR (376 MHz, CD3OD): δ –110.86 (d, JPF = 109 Hz);
31P NMR δ (162 MHz, CD3OD): 2.56 (t, JPF =
109 Hz); HRMS-FAB (m/z): [M+H]+ calcd. for C14H10
[35]Cl2F2NO4P, 394.9692; found,
394.9685.
Inhibitor 2.60.
1H NMR (400 MHz, CD3OD): δ 8.27 (s, 1H), 8.04 (d, J = 8.0 Hz, 2H), 7.99
(d, J = 8.8 Hz, 1H), 7.75 (d, J = 8.0 Hz, 2H), 7.59 (d, J = 8.8 Hz, 1H); 19
F NMR (376 MHz, CD3OD): δ –64.96, –112.45 (d, JPF = 109 Hz);
31P NMR (162 MHz, CD3OD): δ 2.62 (t, JPF =
109 Hz). HRMS-FAB (m/z): [M+H]+ calcd. for C15H11
[35]ClF5NO4P, 430.0034; found, 430.0029.
Inhibitor 2.61.
1H NMR (400 MHz, CD3OD): δ 8.27 (s, 1H), 8.04 (d, J = 8.0 Hz, 2H), 7.92
(d, J = 7.2 Hz, 1H), 7.79–7.74 (m, 3H); 19
F NMR (376 MHz, CD3OD): δ –64.96, –112.46 (d, JPF
= 109 Hz); 31
P NMR (162 MHz, CD3OD): δ 2.62 (t, JPF = 109 Hz). HRMS-FAB (m/z): [M+H]+
calcd. for C15H11[79]
BrF5NO4P, 473.9529; found, 473.9542.
Inhibitor 2.62.
1H NMR (400 MHz, CD3OD): δ 8.42 (s, 2H), 8.07 (d, J = 8.0 Hz, 2H), 7.77
(d, J = 8.0 Hz, 2H), 7.71 (s, 1H); 19
F NMR (376 MHz, CD3OD): δ –65.38, –112.46 (d, JPF = 109
Hz); 31
P NMR (162 MHz, CD3OD): δ 2.54 (t, JPF = 109 Hz). HRMS-FAB (m/z): [M+H]+ calcd.
for C16H11F8NO4P, 464.0297; found, 464.0301.
28
Compound 2.41.
1H NMR (400 MHz, CD3OD): δ 7.43 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0
Hz, 1H), 7.28 (t, J = 7.6 Hz, 2H), 7.22–7.15 (m, 3H), 3.47 (s, 3H); 19
F NMR (376 MHz, CD3OD): δ –112.01 (d, JPF = 109 Hz);
31P NMR (162 MHz, CD3OD): δ 2.62 (t, JPF = 109 Hz).
HRMS-FAB (m/z): [M+2Na–H]+ calcd. for C15H13F2NO4PNa2, 386.0346; found, 386.0354.
General synthesis of other (non-amide) DFMP inhibitors
Free carboxylic acid containing 2.25 was synthesized by subjecting intermediate 2.14 to the
procedure described for deprotection of 2.64. Inhibitors 2.29, 2.33, 2.36, 2.39 and 2.45 were
synthesized as described in the text (vide supra). All compounds were purified either by
automated reversed-phase C18 column chromatography (linear gradient of 5% to 95%
acetonitrile in water with 0.1% trifluoroacetic acid buffer), or by recrystallization.
Analytical data for other DFMP inhibitors
Inhibitor 2.25.
1H NMR (CD3OD): δ 7.93 (d, 2H, J = 7.4 Hz), 7.72 (d, 2H, J = 7.4 Hz);
19F
NMR (CD3OD): δ –110.78 (d, JPF = 111 Hz); 31
PNMR (CD3OD): δ 3.38 (br m). HRMS-ESI
(m/z): [M–H]– calcd. for C8H6O5F2P, 250.9926; found, 250.9916.
Inhibitor 2.29.
1H NMR (400 MHz, CD3OD): δ 7.93 (d, J = 7.6 Hz, 2H), 7.83 (d, J = 8.0 Hz,
2H), 7.61–7.49 (m, 5H); 19
F NMR (376 MHz, CD3OD): δ –111.17 (d, JPF = 113 Hz); 31
P NMR
(162 MHz, CD3OD): δ 3.31 (t, JPF = 113 Hz). HRMS-FAB (m/z): [M+H]+ calcd. for
C14H13F2NO4P, 328.0550; found, 328.0545.
Inhibitor 2.33.
1H NMR (400 MHz, CD3OD): δ 3.74 (2H, s), 7.09 (1H, t, J = 7.4 Hz), 7.30
(2H, t, J = 8.0 Hz), 7.47 (2H, d, J = 8.0 Hz), 7.52–7.56 (2H, m), 7.59 (2H, d, J = 8.0 Hz); 19
F NMR (376 MHz, CD3OD): δ –110.03 (d, JPF = 113 Hz);
31P NMR (162 MHz, CD3OD): δ 4.75
29
(t, JPF = 113 Hz). HRMS-ESI (m/z): [M+H]+ calcd. for C15H15F2NO4P, 342.0701; found,
342.0715.
Inhibitor 2.36.
1H NMR (400 MHz, CD3OD): δ 7.48 (ABq, J = 8.4 Hz, 4H), 7.10 (t, J = 7.8
Hz, 2H), 6.68 (d, J = 8.0 Hz, 2H), 6.64 (t, J = 7.4 Hz, 1H), 4.34 (s, 2H); 19
F NMR (376 MHz,
CD3OD): δ –106.70 (d, JPF = 109 Hz); 31
P NMR (162 MHz, CD3OD): δ 2.78 (t, JPF = 108 Hz).
HRMS-ESI (m/z): [M+H]+ calcd. for C14H15F2NO3P, 314.0758; found, 314.0766.
Inhibitor 2.39.
1H NMR (400 MHz, CD3OD): δ 7.60 (d, J = 8.1 Hz, 2H), 7.47 (d, J = 8.0 Hz,
2H), 7.24 (t, J = 7.9 Hz, 2H), 6.93–6.90 (m, 3H), 5.08 (s, 2H); 19
F NMR (376 MHz, CD3OD): δ
–107.45 (d, JPF = 109 Hz); 31
P NMR (162 MHz, CD3OD): δ 7.01 (br t). HRMS-ESI (m/z):
[M+Na]+ calcd. for C14H13F2O4PNa, 337.0412; found, 337.0415.
Inhibitor 2.45.
1H NMR (400 MHz, CD3OD): δ 7.04–7.13 (m, 3H), 7.19–7.25 (m, 2H), 7.70
(d, J = 8.4 Hz, 2H), 7.85 (d, J = 8.4 Hz, 2H); 19
F NMR (376 MHz, CD3OD): δ –110.80 (d, JPF =
106.0 Hz). MS-ESI (m/z): [M+H]+ calcd. for C13H13F2NO5PS, 364.0142; found, 364.0.
Expression and purification of PtpA
The gene for PtpA was amplified from Mtb genomic DNA and cloned into the pET28b
vector (Novagen). Protein was expressed in BL21(DH3) cells (Invitrogen). Transformed bacteria
were grown to an OD600 of 0.8 in terrific broth and protein expression was induced by the
addition of 100 M isopropyl -D-1-thiogalactopyranoside. After 18 h of expression at 20 °C,
cells were harvested and resuspended in buffer A (20 mM Tris pH 7.5, 50 mM NaCl), and
protease inhibitor AEBSF. Cell suspensions were sonicated, the lysates centrifuged for 1 h at
15,000 × g, and the cleared lysate loaded onto a metal affinity column. After elution with an
imidazole gradient, the His6 tag was cleaved by treatment with 1:1,000 (w/w) trypsin for 10 min
at ambient temperature. The protein was further purified by gel filtration on a S75 Superdex
column in buffer A and concentrated to 2.3 mg/mL for phosphatase assays.
30
Assay procedures
Determination of substrate Km
96-well plates were used with reaction volumes of 100 L per well. 30 L of water was
added to each well, followed by 5 L of buffer (stock solution: 1.0 M Tris-HCl, 20 mM MgCl2,
pH 7.5), 40 L of 2-amino-6-mercapto-7-methylpurine riboside (MESG) solution (stock: 1 mM,
400 M in assay), and 10 L of purine nucleotide phosphorylase (PNP) solution (stock: 0.01
U/mL). 5 L of the appropriate substrate dilution, serially diluted 2.5-fold for a total of 8
different concentrations in DMSO, was then added to the wells, and the plate was covered and
incubated at 37 °C for 5 min in a UV-Vis plate reader. The coupled assay was started by addition
of 10 L of a 1 M stock of PtpA (100 nM in assay), and the reaction progress was monitored at
360 nm at 37 °C. The initial rate data collected was used for Michaelis-Menton kinetic analysis,
where the Km could be obtained. Km and Vmax were determined using nonlinear regression
analysis on the substrate-velocity data with the equation v = Vmax*[S]/(Km+[S]).
Determination of inhibitor Ki
96-well plates were used with reaction volumes of 100 L per well. 45 L of water was
added to each well, followed by 20 L of sodium citrate buffer (stock solution: 100 mM sodium
citrate, pH 6.2, 0.02% Triton X-100), 5 L of 20 mM EDTA stock solution (1 mM in assay), 5
L of 20 mM DTT stock solution (1 mM in assay), and 10 L of 1 M PtpA stock solution (100
nM in assay). Then 5 L of the appropriate inhibitor stock solutions, serially diluted 2-fold for a
total of 10 different concentrations in DMSO, was added to the wells, and the plate was covered
and incubated for 5 min at 37 °C in a UV-Vis plate reader. The reaction was started by addition
of 10 L of 2 mM pNPP substrate stock (200 M in assay), and reaction progress was monitored
at 405 nm with continued incubation at 37 °C. The initial rate data collected was used for the
determination of Ki values. The kinetic values were obtained from nonlinear regression of
substrate-velocity curves in the presence of various concentrations of inhibitor using the equation
v = Vmax*[S]/(Km(1+[I]/Ki)+[S]).
Inhibitor-PtpA modeling
Receptor relaxation
The X-ray crystal structure of PtpA (PDB ID 1U2P) was used for all modeling studies.19
To
allow the protein to relax, a short molecular dynamics simulation was run in AMBER 9.0 using
the ff03 force field.20,24
The structure was prepared by removing crystallographic waters and
adding an 8.0 Å octagon of TIP3P water and sodium ions using the LEaP accessory.24
The
system was minimized, slowly melted to 300K, and allowed to equilibrate for 25 ps. The
simulation was continued for an additional 150 ps and selected as the final snapshot for docking.
Active site identification
To identify where inhibitors might bind, water molecules were removed from the structure
produced by the molecular dynamics simulation, and protein surface invaginations were
31
identified using spheres generated by the DOCK accessory SPHGEN.25
The putative binding site
was characterized by selecting all spheres within a 12 Å radius of the chlorine atom bound to the
active-site cysteine nucleophile in the X-ray structure.
Receptor preparation for docking
An octagon of TIP3P waters was built around the receptor using the Chimera AmberTools
module, followed by removal of any water molecules >5 Å from any receptor atom, resulting in
approximately two shells of water molecules.26
All waters <3 Å from the active site spheres
described above were then removed. The Chimera (version 1.3) Dock Prep module was used to
complete the receptor preparation.27
To account for the receptor contribution to the score during
DOCKing, grids were precomputed to store the van der Waals and electrostatic values for the
receptor using the DOCK accessory GRID.27a
Compound preparation for docking
To validate observed structure-activity relationships, structures 2.09 and 2.63 were docked
onto PtpA. Each compound was drawn and converted to SMILE strings using the JME molecular
editor.28
The SMILE strings were used to create rotamer ensembles of three-dimensional
structures in OMEGA.21
All generated conformations were kept for docking, resulting in an
average of 11 conformations per compound. Each conformation was protonated and assigned
AM1-BCC charges using the Chimera (version 1.3) AddH and AddCharge modules.29
Docking procedure
The compound conformations were docked using Grid Score in DOCK 6.4 using default
parameters.21
Each conformation was then rescored and ranked using the PB/SA score. The top
scoring conformation for each compound was used for comparisons. For compounds 2.09 and
2.63, molecular dynamics simulations were also performed on the top-scoring conformation to
explore the validity of the docked poses. The same protocol described in the Receptor Relaxation
section was used. Both simulations were equilibrated after 100 ps, and the simulations were run
for a further 50 ps. The snapshot with the ligand heavy atom RMSD closest to the docked pose
from the final 50 ps of each simulation was selected for analysis.
References
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X.-W.; Wang, S.; Wu, L.; Zhang, Z.-Y.; Burke Jr., T. R. Bioorg. Med. Chem. 1998, 6, 1799; (d)
Puius, Y. A.; Zhao, Y.; Sullivan, M.; Lawrence, D. S.; Almo, S. C.; Zhang Z.-Y. Proc. Natl.
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12. Han, Y.; Belley, M.; Bayly, C. I.; Colucci, J.; Dufresne, C.; Giroux, A.; Lau, C. K.; Leblanc,
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52, 2067; (b) Shoichet, B. J. Med. Chem. 2006, 49, 7274; (c) Shoichet, B. Drug Discov. Today
2006, 11, 607; (d) Feng, B. Y.; Shoichet, B. K. Nat. Protoc. 2006, 1, 550; (e) Seidler, J.;
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C. B.; Iversen, L. F. Handb. Exp. Pharmacol. 2005, 167, 215; see also reference 5a.
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Andersen, O. A.; Rao, F. V.; Allwood, M.; Lloyd, C.; Eggleston, I. M.; van Aalten, D. M. F. J.
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Structure 1999, 7, 297.
23. Molecular graphics images were produced using the UCSF Chimera package from the
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Francisco (supported by NIH P41 RR-01081).
24. Duan, Y.; Wu, C.; Chowdhury, S.; Lee, M. C.; Xiong, G.; Zhang, W.; Yang, R.; Cieplak, P.;
Luo, R.; Lee, T.; Caldwell, J.; Wang, J.; Kollman, P. J. Comput. Chem. 2003, 24, 1999.
25. Lee, M. C.; Duan, Y. Proteins 2004, 55, 620.
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Phys. 1983, 79, 926.
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Langridge, R.; Ferrin, T. E. J. Mol. Biol. 1982, 161, 269.
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R. C. J. Comput.-Aided Mol. Des. 2006, 20, 601.
29. Shoichet, B. K.; Bodian, D. L.; Kuntz, I. D. J. Comput. Chem. 1992, 13, 380.
34
35
Chapter 3. Fragment-based identification of inhibitors of striatal-enriched protein tyrosine
phosphatase
Abstract: In this chapter, the brain-specific phosphatase, STriatal-Enriched protein tyrosine
Phosphatase (STEP) is introduced as a therapeutic target for neurodegenerative diseases. The
substrate-based fragment screening approach from chapters 1 and 2 was used to identify lead
compounds for STEP inhibition. Through this fragment-based approach, we were able to identify
many low molecular weight (<450 Da), nonpeptidic, single-digit micromolar mechanism-based
STEP inhibitors with greater than 20-fold selectivity across multiple human PTPs and DUSPs.
Additionally, significant levels of STEP inhibition in rat cortical neurons were also observed for
our most potent inhibitors. The majority of this work has been published as a full article
(Baguley, T. D.; Xu, H.-C.; Chatterjee, M.; Nairn, A. C.; Lombroso, P. J.; Ellman, J. A. J. Med.
Chem. 2013, 56, 7636).
36
Authorship
This work was conducted in collaboration with Dr. Hai-Chao Xu and Dr. Manavi Chatterjee.
Dr. Xu and I synthesized the inhibitor library. I expressed and purified the enzyme STEP for
assays. The substrate and inhibitor assays were performed by Dr. Xu and myself. Dr. Chatterjee
performed all cell based assays. Blood-brain barrier permeability assays were performed by Pion,
Inc. (Billerica, MA).
Introduction
Synaptic connections provide the physical basis for communication within the brain, and
synaptic plasticity, the ability for synapses to strengthen or weaken between neurons as a result
of molecular signals, is critical to maintaining proper cognitive function. Therefore, disruptions
in synaptic function can lead to impairments in cognition. Synaptic dysregulation has been
implicated in a range of neurodegenerative diseases,1 including Alzheimer’s disease (AD),
2
schizophrenia,3 depression,
4 fragile X syndrome,
5 and drug addiction.
6
One protein that has been implicated in the dysregulation of synaptic plasticity is STriatal-
Enriched protein tyrosine Phosphatase (STEP), which is found in striatum, hippocampus, cortex,
and related regions of the brain. High levels of STEP activity result in the dephosphorylation and
inactivation of several neuronal signaling molecules including extracellular signal-regulated
kinases 1 and 2 (ERK1/2),7 proline-rich tyrosine kinase 2 (Pyk2),
8 mitogen activated protein
kinase p38,9 and the GluN2B subunit of the N-methyl-D-aspartate receptor (NMDAR).
10,11
Dephosphorylation of the kinases inactivates them, while dephosphorylation of GluN2B results
in internalization of NMDA receptors. To test the hypothesis that overexpression of STEP might
contribute to cognitive deficits in AD mouse models, STEP levels were reduced genetically in
AD mice. Progeny null for STEP exhibited significant cognitive improvements and increased
receptor levels on synaptic membranes.11
These results support STEP as a potential target for
drug discovery for the treatment of AD.
As described in chapter 1, the Ellman group has developed a substrate-based fragment-
screening approach to identify small molecule inhibitors of phosphatases termed substrate
activity screening (SAS). In chapter 2, this method was used to identify inhibitors of PtpA from
Mtb. Prior to our work on STEP, there was a single reported ancillary example of a STEP
inhibitor having modest activity and without selectivity or cell data.12
In this chapter, I will
discuss the application of the SAS method to identify low molecular weight (<450 Da)
nonpeptidic STEP inhibitors with single-digit micromolar inhibition, 20-fold selectivity over
multiple human PTPs and DUSPs, and significant activity in rat cortical neurons.
Inhibitor scaffold identification
Identification of inhibitors for STEP began with screening the same previously synthesized
O-aryl phosphate library as was used in chapter 2.13
Initially, there were several promising
fragment substrates (Figure 3.1). Substitution ortho to the phosphate was not well tolerated, but
meta and para substitution seemed to be allowed. Of note, fragment substrates 3.06–3.10 had
much improved Km values relative to the protected phosphotyrosine derivative 3.04, which more
closely resembles the natural phosphotyrosine in PTP substrates.
37
Figure 3.1. Selected initial substrate hits obtained against STEP.
Because of the ease of synthesis of amide containing aryl phosphates (chapter 2, Scheme
2.1), we were first drawn to scaffold 3.09. However, after much effort to optimize the scaffold
through substitution of the aniline ring as was carried out for Mtb PtpA as described in chapter 2,
synthesizing and testing > 50 novel substrates, no substrate gave a more potent lead than the
completely unsubstituted aniline (Figure 3.2). At this point, we decided to identify a non-
hydrolyzable phosphate mimetic that would provide inhibitory activity versus STEP (Figure 3.3).
Upon assaying the DFMP14
(3.12), isothiazolidinone15
(3.13) and isoxazole carboxylic acid16
(3.14) inhibitors, it was apparent that only the DFMP pharmacophore was active for inhibition.
Figure 3.2. Attempted optimization of substrate fragment 3.09 was unsuccessful.
38
Figure 3.3. Conversion of substrate 3.09 into inhibitors by replacement of the phosphate with non-
hydrolyzable mimics demonstrates that DFMP is the only active pharmacophore.
It was at this point that we went back to our original substrate screening results (Figure 3.1).
We then identified 3.06 and 3.08 for further optimization because the biphenyl scaffold has been
regarded as a “privileged scaffold” with druglike properties and because analog preparation is
straightforward using cross-coupling methodology.17
Inhibitors 3.15 and 3.16 (Figure 3.4) were
prepared by replacing the phosphate group with the DFMP isostere, and, gratifyingly, the Ki
values of the inhibitors correlated reasonably well with the Km values of their corresponding
substrates,18
which is desired for substrate analog inhibitors.
Figure 3.4. DFMP inhibitors 3.15 and 3.16 based on privileged substrate scaffolds 3.06 and 3.08.
The biaryl DFMP inhibitors could be conveniently prepared by relying on the Suzuki-
Miyaura cross-coupling reaction (Scheme 3.1). The commercial availability of many diverse
arylboronic acids and boronic esters (ArBLn) enabled rapid access to an initial set of biaryl
inhibitors from the known aryl bromides 3.21 and 3.22.13b,18b
However, for the further
optimization of the inhibitor structures we needed to prepare the requisite more complex
arylboronic acid inputs, which will be the bulk of the synthetic discussion in this chapter.
39
Scheme 3.1. Inhibitor synthesis through a Suzuki-Miyaura cross-coupling strategy
Optimization of inhibitor 3.15
After an initial inhibitor library screen for the inhibitors based on the 3-biphenyl scaffold
(Table 3.1), some trends started to become apparent. Although alkyl substitution at the para
position of the distal ring was beneficial for inhibition (3.39), any substitution larger than a
methyl group (3.38), or inclusion of electron deficient halogens (3.30, 3.36) or electron donating
heteroatoms (3.24–3.25, 3.27–3.28) was met with decreased potency. Likewise, alkyl
substitution at the meta position also led to an increase in potency of the inhibitors, with the -
branched and more bulky isopropyl group (3.43) outperforming the methyl group (3.42).
However, replacement of the alkyl group at the meta position with electron-rich (3.26, 3.29,
3.35, 3.40) or electron-withdrawing (3.32, 3.34) groups was not as beneficial as alkyl
substitution (3.42–3.43). The presence of a hydroxyl group at the ortho position was also
beneficial to the potency of the inhibitors (3.41), but not at the meta (3.29) or para (3.27)
position. This could be due to H-bond donor ability, as the methylated version (3.31) and
halogens (3.33, 3.37) that can accept H-bonds through their lone pairs, all suffered in potency
relative to the free hydroxyl.
At this point, we wanted to explore the additive effects of the ortho hydroxyl (3.41) and meta
alkyl (3.43) groups. Because these more complicated aryl boronic acids were not commercially
available, they had to be synthesized. When the desired alkyl phenols were commercially
available, the 2-hydroxyphenylboronic acids were synthesized by a two-step sequence (Scheme
3.2). First, selective bromination of the phenol (3.44) was accomplished by NBS in the presence
of a catalytic amount of N,N-diisopropylamine.19
The bromo compound was then dilithiated and
treated with trimethyl borate to yield the boronic acids (3.46) after aqueous workup.20
In most
cases, the boronic acid was used crude in the Suzuki-Miyaura cross-coupling reaction, and
although by NMR analysis it appeared that there were multiple species in equilibrium, likely
anhydrides between the hydroxyls on both the aryl rings and the boron centers, by LCMS the
crude products contained a single peak corresponding to the boronic acid mass in all cases.
When the desired alkyl phenols were not commercially available, they had to be synthesized
prior to borylation (Scheme 3.3). This was accomplished through a dilithiation of ortho-
bromophenol and quenching with an aldehyde or ketone. The resulting benzyl alcohols (3.48)
were then treated with triethylsilane (Et3SiH) under acidic conditions to reductively eliminate the
alcohol to yield the requisite 2-alkyl phenols (3.49). While these alkylphenols could be subjected
to the bromination-borylation sequence in Scheme 3.2, in order to increase the efficiency of the
sequence, they were instead subjected to an iridium-catalyzed one-pot silyl-directed ortho-
borylation disclosed by Hartwig and coworkers. Subsequent treatment with potassium hydrogen
difluoride (KHF2) then provided the trifluoroborates 3.50.21
40
Table 3.1. Representative examples from initial 3-biphenyl DFMP inhibitor screening against STEPa
cpd Ar Ki (M) cpd Ar Ki (M) cpd Ar Ki (M)
3.15
337 ± 60 3.30
468 ± 39 3.37
233 ± 23
3.24
>500 3.31
271 ± 17 3.38
222 ± 1
3.25
>500 3.32
267 ± 17 3.39
205 ± 1
3.26
>500 3.33
260 ± 32 3.40
177 ± 8
3.27
>500 3.34
259 ± 53 3.41
168 ± 17
3.28
>500 3.35
240 ± 13 3.42
137 ± 8
3.29
486 ± 2 3.36
238 ± 14 3.43
81 ± 2
aKi values were determined using at least two independent measurements.
Scheme 3.2. Synthesis of arylboronic acids from 2-alkylphenols
Once synthesized, the inhibitors were screened against STEP (Table 3.2). Combining the
meta isopropyl group and the ortho hydroxyl group in a 2,3-orientation (3.51) led to an increase
in potency, while the 2,5-substitution was not advantageous (3.52). We next investigated the
effect of altering the meta alkyl group (3.53–3.60) on inhibitory potency. The ethyl group (3.53)
resulted in 2-fold reduction in inhibitory potency, while the tert-butyl group provided a modest
41
Scheme 3.3. Synthesis of aryltrifluoroborates from 2-bromophenol
enhancement (3.54). Branching at the -carbon was confirmed to be important when comparing
the n-propyl and isobutyl compounds (3.55–3.56). Knowing that -branching was important, we
discovered that a more significant increase in potency was achieved by introducing cycloalkyl
groups (3.57–3.60), with cyclohexyl (3.59) providing the optimal ring size (Ki = 20 ± 3 M).
Finally, introduction of the -hydroxymethylphosphonic acid phosphate mimetic (3.61, Figure
3.5) in place of difluoromethylphosphonic acid resulted in an approximate 2-fold reduction in
potency.
Table 3.2. Screening of focused 3-biphenyl DFMP inhibitors against STEP
a
cpd Ar Ki (M) cpd Ar Ki (M) cpd Ar Ki (M)
3.41
168 ± 17 3.53
124 ± 3 3.57
45 ± 2
3.43
81 ± 2 3.54
63 ± 1 3.58
25 ± 4
3.51
69 ± 9 3.55
70 ± 3 3.59
20 ± 3
3.52
95 ± 1 3.56
53 ± 1 3.60
62 ± 1
aKi values were determined using at least two independent measurements.
42
Figure 3.5. Replacement of the DFMP pharmacophore with an -hydroxyphosphonic acid was met with
diminished potency, despite the results from the 4-biphenyl scaffold (vide infra).
Optimization of inhibitor 3.16
A separate inhibitor library based on 4-biphenyl DFMP inhibitor 3.16 was synthesized
utilizing the Suzuki-Miyaura cross-coupling sequence (Scheme 3.1) and was screened for
activity against STEP (Table 3.3). Electron deficient (3.63, 3.65, 3.66), donating (3.67, 3.68) and
even alkyl (3.69, 3.70) groups all proved detrimental to inhibitor potency. Borrowing
information from compound 3.41, we next tried to introduce H-bond donors, and found that they
were detrimental at the ortho (3.62) and para (3.64) positions, but moderately increased inhibitor
potency when placed at the meta position (3.71–3.73), with benzyl alcohol 3.73 providing the
largest increase in potency. However, the greatest potency was observed for benzyl substitution
Table 3.3. Representative examples from initial 4-biphenyl DFMP inhibitor screening against STEP
a
cpd Ar Ki (M) cpd Ar Ki (M) cpd Ar Ki (M)
3.16
120 ± 7 3.66
375 ± 30 3.71
113 ± 1
3.62
>500 3.67
231 ± 6 3.72
112 ± 9
3.63
>500 3.68
200 ± 1 3.73
88 ± 3
3.64
450 ± 183 3.69
185 ± 33 3.74
73 ± 3
3.65
400 ± 48 3.70
132 ± 3
aKi values were determined using at least two independent measurements.
43
at the meta position (3.74), which resulted in a near 2-fold enhancement over unsubstituted 3.16.
With this information in hand, we turned toward synthesizing inhibitors with varying
substituents on the aryl ring of the benzyl substituent of inhibitor 3.74. The boronic acid coupling
partners needed for the inhibitor syntheses (Scheme 3.4) were generated first by the addition of
in situ generated 3-bromolithium to a variety of aldehydes (3.75) to yield diarylmethanols, 3.76.
After reductive elimination of the benzyl alcohol, the aryl bromide was converted to the pinacol
boronic ester 3.78 with a Miyaura borylation.22
Scheme 3.4. Synthesis of diarylmethane-based boronic esters for the synthesis of 3.74 analogs
After screening compounds with a variety of substituents on the benzyl ring, it was
discovered that electron deficient halogens were most beneficial for binding (Figure 3.6). Other
substituents were tried (alkyl, alkoxy, etc.; data not shown), but the halogens showed the greatest
increase in potency relative to compound 3.74 (Ki = 73 ± 3 M). The ideal substitution pattern
was the 3,4-dichlorobenzyl inhibitor 3.81 (Ki = 9.6 ± 0.6 M), but the compound had very
limited solubility in aqueous media. The hydroxyl group, which was previously observed to be
well tolerated at the benzylic position (see 3.73, Table 3.3), increased the solubility of the
inhibitor with only a moderate reduction in potency (3.82). Replacing the DFMP pharmacophore
with an -hydroxyphosphonic acid group maintained the desired potency (3.83) as well as
increased the aqueous solubility.
Figure 3.6. Selected screening results versus STEP from the initial focused benzyl inhibitor library.
The -hydroxymethylphosphonic acid inhibitor 3.83 was also prepared by a Suzuki-Miyaura
cross-coupling reaction (Scheme 3.5). Ketone 3.86 was obtained by cross-coupling
ketophosphonic acid 3.8423
with arylboronic acid derivative 3.85, and sodium borohydride
reduction then led to the final -hydroxymethylphosphonic acid compound.
44
Scheme 3.5. Synthesis of -hydroxyphosphonic acid inhibitor 3.83
We next explored the effect of combining the hydroxyl groups in inhibitors 3.82 and 3.83 by
the preparation of the four possible stereoisomers, 3.97–3.100 (Scheme 3.6). The four
stereoisomeric inhibitors were prepared from the enantiomerically pure -hydroxymethyl-
phosphonic acids 3.90 and 3.91 and the enantiomerically enriched boronic esters 3.95 and 3.96
by Suzuki-Miyaura cross-coupling. The synthesis of the enantiomerically pure phosphonic acids
started with the addition of tris-[(1S,2R,5S)-menth-2-yl]phosphite to 4-bromobenzaldehyde to
give a mixture of diastereomers 3.88 and 3.89, which were separated by recrystallization
according to literature procedures for analogous compounds.24
Removal of the menthyl groups
by treatment with TMSCl and NaI afforded phosphonic acids 3.90 and 3.91. The absolute
stereochemistry of the -hydroxyphosphonic acids was confirmed by chemical correlation
through hydrodebromination of 3.91 to the corresponding -hydroxyphenylmethylphosphonic
acid, for which the absolute configuration had previously been determined.25
The catalytic
asymmetric addition of a 3,4-dichlorophenylzinc reagent to 3-bromobenzaldehyde using each
enantiomer of 3-exo-(morpholino)isoborneol (MIB)26
gave the enantiomerically enriched
diarylmethanols 3.93 and 3.94 in 90% ee. Subsequent Miyaura borylation led to the boronic
esters 3.95 and 3.96.22
45
Scheme 3.6. Synthesis of the four stereoisomeric -hydroxymethylphosphonic acid inhibitors 3.97–3.100
With the diastereomerically pure inhibitors in hand, we were able to test their potency against
STEP (Figure 3.7) While the potency is not affected by the stereochemistry of the distal
biarylmethanol (3.97 versus 3.98), the (S)-configuration at the -hydroxyphosphonic acid is
crucial for inhibitory activity. Further evidence of the importance of this particular orientation of
the hydroxyl group was that the (R)-configuration (3.99 and 3.100) provided no better inhibition
than when the hydroxyl was removed completely (3.101).
Figure 3.7. Screening results for diastereomerically pure inhibitors 3.97–3.100 against STEP.
46
Inhibitor selectivity profile
As mentioned in chapter 1, achieving selectivity for PTP inhibition is very challenging due to
the high structural homology among PTP active sites.27
Additionally, knowing the selectivity
needed for therapeutic relevance is very challenging because the therapeutic window is
dependent on a variety of independent factors that are difficult or impossible to predict including
severity of side effects and redundancy in signaling cascades. A drug compound may be
sufficient with 10-fold selectivity, or may require 1000-fold selectivity, but when dealing with
challenging targets such as PTPs, this information is often not known without some first-in-class
inhibitors to use as tool compounds. The best inhibitors from the two biaryl series, compounds
3.59, 3.97, and 3.98, were tested against a panel of PTPs and DUSPs (Table 3.4). Notably, our
most potent inhibitors from the 1,4-biphenyl series 3.97 and 3.98 displayed greater than 20-fold
selectivity against all phosphatases tested, whereas compound 3.59 from the 1,3-biphenyl series
displayed more modest selectivity. These were the first small molecule inhibitors of STEP
reported in the literature with any selectivity data.
Table 3.4. Selectivity profile of inhibitors 3.59, 3.97 and 3.98 against a panel of human PTPs
a
STEP TC-Ptp CD45 LAR MKP5
3.59 Ki, M 20 ± 3 71 ± 2 >500 >900 150 ± 20
selectivity -- 3.6 >25 >45 7.5
3.97 Ki, M 8.9 ± 1.2 164 ± 9 340 ± 40 360 ± 30 340 ± 45
selectivity -- 18 38 40 41
3.98 Ki, M 7.8 ± 0.7 170 ± 20 270 ± 20 390 ± 110 >500
selectivity -- 22 35 50 >65 aKi values were determined using at least two independent measurements.
STEP inhibition in neuronal cultures
The most selective inhibitors, 3.97 and 3.98, were also evaluated for their ability to inhibit
STEP in rat cortical neurons by monitoring the phosphorylation levels of the known STEP
substrates GluN2B, Pyk2 and ERK1/2.7,8,10,11
Clear increases in the phosphorylation levels of
each of the substrates were observed for inhibitor 3.97 with more modest effects observed for
3.98 (Figure 3.8) indicating that the deposphorylation activity of STEP was inhibited. Selectivity
for non-STEP PTP substrate proteins was not assessed in this study.
Blood-brain barrier permeability
Given the promising cell data, the best compound, 3.97, was evaluated for its ability to
passively permeate the blood-brain barrier (BBB) using the well validated parallel artificial
membrane permeability assay (PAMPA) technique.28
In this assay, the compound of interest is
added to a donor well and its ability to passively cross a membrane mimic is assessed through
detection of the compound in an acceptor well which is separated from the donor well only by
the membrane mimic. Although the compound can cross cell membranes, as evidenced in the
above cell data, compound 3.97 did not possess the ability to passively cross the BBB in this
model system (Table 3.5). BBB-PAMPA studies were conducted by Pion, Inc (Billercia, MA) at
47
pH 7.4. The refined effective permeability (Pe) value obtained (average of triplicates) is
summarized in the table along with results for internal highly and low permeable standards,
propranolol and atenolol respectively.
Figure 3.8. Rat cortical neurons were treated with vehicle or 3.97 (a) or 3.98 (b) (concentrations of 0.1, 1 or 10 µM) for 1 h and analyzed by Western blotting. (*p < 0.05; **p < 0.01; ***p < 0.001 one-way
ANOVA, Dunnett’s post hoc). Data represent the phospho-signal normalized to the total substrate protein
signal and GAPDH as a basic expression level control + s.e.m. (n = 3–5 each group).
Table 3.5. BBB-PAMPA assay results for compound 3.97
Compound Avg. Pe
(10–6
cm/s)a Avg. %R
b Avg. logPe Domain, nm
Inhibitor 3.97 <0.1 4 ± 0 260–350
propranolol 68 ± 5 48 ± 3 –4.17 ± 0.03 250–498
atenolol <0.4 1 ± 1 250–498
aeffective permeability measured in assay; results indicated with a “<” sign mean no quantifiable UV
signal was detected in the acceptor compartments. bmembrane retention.
With these results, BBB permeability remains a real challenge for future work. The highly
polar nature of the -hydroxyphosphonic acid motif is likely to be one of the key factors limiting
BBB permeability. Replacement of this motif with less polar, non-hydrolyzable phosphate
mimetics might overcome this problem.29
Alternatively, effective prodrug strategies have also
been developed to mask polar functionality in order to enable BBB permeability.30
Conclusions
Although high STEP activity has been observed in many neuropsychiatric disorders, such as
Alzheimer’s disease, prior to this work there had been no selective and potent inhibitors reported
for potential treatment and study of these diseases. The work in this chapter describes the first
dedicated effort for the identification of selective small-molecule mechanism-based inhibitors of
48
STEP. A library of low molecular weight O-aryl and -heteroaryl phosphate fragments were
screened, which identified both the 4- and the 3-biaryl inhibitors (3.16 and 3.15 respectively) as
promising templates for inhibitor development. SAR of the scaffolds was explored to identify
potent inhibitors from each series, with the most potent inhibitors (3.59, 3.97, and 3.98) all
showing promising selectivity over other human phosphatases tested. Importantly, the most
selective inhibitors 3.97 and 3.98 were able to inhibit STEP in rat cortical neurons as indicated
by the significant increase in phosphorylation levels of STEP substrates.
Experimental
General synthetic methods
Unless otherwise noted, all reagents were obtained from commercial suppliers and used
without further purification. Tetrahydrofuran (THF), dioxane, CH2Cl2, and diethyl ether (Et2O)
were passed through a column of activated alumina (type A2, 12 × 32, Purify Co.) under
nitrogen pressure immediately prior to use. All 1H,
19F, and
31P NMR spectra were obtained at
ambient temperature on a Bruker AVB-400 or AVB-500 spectrometer. NMR chemical shifts are
reported in ppm relative to TMS (0.00), CHCl3 (7.26), acetone (2.05) or CH3OH (3.31) for 1H,
trifluoroacetic acid (−76.55) for 19
F, and H3PO4 (0.00) for 31
P. Mass spectrometry (HRMS, ESI)
are reported in m/z. Chromatography was performed either with SiliCycle SiliaFlash P60
230−400 mesh silica gel or by utilizing a Biotage SP1 flash purification system (Biotage model
SP1-B1A) or a Teledyne Isco CombiFlash Rf system. Reversed-phase purifications were
conducted with a Teledyne Isco CombiFlash Rf system equipped with HP C18 gold cartridges.
Product yields are not optimized. Enzymatic assays were carried out on a BioTek Synergy 2
multimode microplate reader. All of the tested substrates and inhibitors displayed ≥ 95% purity
as determined by HPLC or UPLC. BBB PAMPA studies were conducted by Pion, Inc. (Billerica,
MA).
Synthesis of and analytical data for biphenyl inhibitors
Compound 3.21. Iodotrimethylsilane (5.22 mL, 36.6 mmol, 2.2 equiv) was added to a stirred
solution of diethyl compound 3.19 (5.71 g, 16.6 mmol) (synthesized as per a previous report,18b
see Scheme 3.1 for more details) in CH2Cl2 (50 mL, 0.3 M). The reaction solution was stirred at
ambient temperature for 14 h. The volatile components were then removed by a rotary
evaporator under vacuum. Sodium hydroxide (664 mg, 16.6 mmol) in methanol (20 mL) was
added to the resulting residue, and this solution was stirred at ambient temperature for 1 h,
allowing the monosodium salt of the desired compound to precipitate from the solution. The
solvent was removed by filtration. LCMS showed the presence of an undesired byproduct in the
collected crude product; thus, it was purified via reversed-phase gradient column
chromatography (5−100% acetonitrile in water with 0.1% trifluoroacetic acid buffer). Volatile
components were removed, and the resulting water solution was lyophilized to afford the product
49
as an off-white powder (2.75 g, 9.44 mmol, 57%). 1H NMR (400 MHz, CD3OD): δ 7.74 (s, 1H),
7.67 (d, J = 8.0 Hz, 1H), 7.58 (d, J = 7.8 Hz, 1H), 7.41 (t, J = 7.9 Hz, 1H); 19
F NMR (376 MHz, CD3OD): δ −110.13 (d, JPF = 110 Hz);
31P NMR (162 MHz, CD3OD): δ 6.76 (br). HRMS-ESI
(m/z): [M−H]–
calcd. for C7H5[79]
BrF2O3P, 284.9133; found, 284.9129.
Compound 3.22. Compound 3.22 was synthesized similarly to 3.21, and is a known
compound. Analytical data matched the previous report.13b
Synthesis of and analytical data for DMFP inhibitors of the general form 3.23
Synthesis of biaryl inhibitors was accomplished through a Suzuki-Miyaura cross-coupling
from either compound 3.21 or 3.22. Two different procedures were employed.
General synthesis of inhibitors by Suzuki-Miyaura cross-coupling (A). A 1 dram oven-dried
vial was charged with a stir bar, the appropriate aryl bromide (1.0 equiv), organoboron species
(1.5 equiv), potassium carbonate (5 equiv), palladium(II) chloride or palladium(II) acetate (5 mol
%), and (2-biphenyl)dicyclohexylphosphine (10 mol %). After addition of all reagents, solvent
(4:1 dioxane/water) was added to the vial (0.25 M in aryl bromide). The vial was sealed with a
screw top containing a Teflon septum and placed in a preheated heating block at 80 °C, and the
mixture was vigorously stirred for 18 h. The vial was then removed from the heating block and
allowed to cool to ambient temperature, followed by addition of 0.3 volumes of 10 N HCl open
to air. The reaction mixture was diluted with 1 volume of water and 1 volume of methanol,
filtered through a Kimwipe, and purified by reversed-phase gradient column chromatography
(5−100% acetonitrile in water with 0.1% trifluoroacetic acid). Volatile components were
removed, and the resulting water solutions were lyophilized to afford the products.
General synthesis of inhibitors by Suzuki-Miyaura cross coupling (B). A 1 dram oven-dried
vial was charged with a stir bar, the appropriate aryl bromide (1.0 equiv), organoboron species
(1.5 equiv), sodium carbonate (6 equiv), and tetrakis(triphenylphosphine)palladium(0) (10 mol
%). After addition of all reagents, solvent (4:1:1 dimethoxyethane/ethanol/water) was added to
the vial (0.10−0.25 M in aryl bromide). The vial was sealed with a screw top containing a Teflon
septum and placed in a preheated heating block at 80 °C to stir for 4−18 h with vigorous stirring.
The vial was then removed from the heating block and allowed to cool to ambient temperature,
followed by addition of 0.3 volumes of 10 N HCl open to air. The reaction mixture was diluted
50
with 1 volume of water and 1 volume of methanol, filtered through a Kimwipe, and purified by
reversed-phase gradient column chromatography (5−100% acetonitrile in water with 0.1%
trifluoroacetic acid). Volatile components were removed, and the resulting water solutions were
lyophilized to afford the products.
Biphenyl inhibitors 3.15–3.16, 3.24–3.43, and 3.62–3.74 were synthesized following one of
the two general procedures described for the Suzuki-Miyaura cross-coupling (general procedure
A or general procedure B) from aryl bromide 3.21 or 3.22. Analytical data for inhibitors 3.15 and
3.16 match previous literature reports.31,13b
Analytical data for inhibitors 3.24–3.43, and 3.62–3.74
Inhibitor 3.24.
1H NMR (400 MHz, CD3OD): 7.69 (s, 1H), 7.59 (d, J = 6.8 Hz, 1H), 7.49–
7.36 (m, 4H), 6.88 (d, J = 8.7 Hz, 2H), 4.53 (hept, J = 6.0 Hz, 1H), 1.22 (d, J = 6.0 Hz, 6H); 19
F
NMR (376 MHz, CD3OD): –110.79 (d, JPF = 113 Hz); 31
P NMR (162 MHz, CD3OD): 7.22
(t, JPF = 113 Hz). MS-ESI (m/z): [M−H]− calcd. for C16H16F2O4P, 341.08; found, 341.1.
Inhibitor 3.25.
1H NMR (400 MHz, CD3OD): 7.92 (s, 1H), 7.65–7.60 (m, 6H), 7.45 (t, J =
7.8 Hz, 1H), 2.14 (s, 3H);19
F NMR (376 MHz, CD3OD): –108.45 (d, JPF = 98 Hz); 31
P NMR
(162 MHz, CD3OD): 6.02 (t, JPF = 98 Hz). HRMS-ESI (m/z): [M+H]+ calcd. for
C15H15F2NO4P, 342.0701; found, 342.0697.
Inhibitor. 3.26.
1H NMR (400 MHz, CD3OD): 7.74 (s, 1H), 7.64 (d, J = 7.5 Hz, 1H), 7.54–
7.39 (m, 4H), 7.34–7.24 (m, 2H), 2.05 (s, 3H); 19
F NMR (376 MHz, CD3OD): –110.90 (d, JPF
= 113 Hz); 31
P NMR (162 MHz, CD3OD): 6.95 (t, JPF = 113 Hz). HRMS-ESI (m/z): [M+H]+
calcd. for C15H15F2NO4P, 342.0701; found, 342.0694.
51
Inhibitor 3.27.
1H NMR (400 MHz, CD3OD): 7.80 (s, 1H), 7.73–7.66 (m, 1H), 7.56–7.47
(m, 4H), 6.90 (d, J = 8.6 Hz, 2H); 19
F NMR (376 MHz, CD3OD): –110.83 (d, JPF = 114 Hz); 31
P NMR (162 MHz, CD3OD): 7.12 (t, JPF = 114 Hz). MS-ESI (m/z): [M−H]− calcd. for
C13H10F2O4P, 299.03; found, 299.1.
Inhibitor 3.28.
1H NMR (400 MHz, CD3OD): 7.70 (s, 1H), 7.60 (d, J = 6.6 Hz, 1H), 7.48
(d, J = 8.7 Hz, 2H), 7.45–7.38 (m, 2 H), 6.92 (d, J = 8.7 Hz, 2H), 3.74 (s, 3H); 19
F NMR (376
MHz, CD3OD): –110.83 (d, JPF = 113 Hz); 31
P NMR (162 MHz, CD3OD): 7.12 (t, JPF = 113
Hz). HRMS-ESI (m/z): [M+H]+ calcd. for C14H14F2O4P, 313.0592; found, 313.0573.
Inhibitor 3.29.
1H NMR (400 MHz, CD3OD): 7.83 (s, 1H), 7.74 (d, J = 7.4 Hz, 1H), 7.64–
7.51 (m, 2H), 7.30 (t, J = 7.9 Hz, 1H), 7.12 (d, J = 7.8 Hz, 1H), 7.08 (t, J = 2.1 Hz, 1H), 6.83 (dd,
J = 8.1, 2.2 Hz, 1H); 19F NMR (376 MHz, CD3OD): –110.90 (d, JPF = 113 Hz); 31
P NMR (162
MHz, CD3OD): 7.03 (t, JPF = 113 Hz). MS-ESI (m/z): [M−H]− calcd. for C13H10F2O4P, 299.03;
found, 299.1.
52
Inhibitor 3.30.
1H NMR (400 MHz, CD3OD): 7.70 (s, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.55
(dd, J = 8.7, 5.3 Hz, 2H), 7.51–7.40 (m, 2H), 7.09 (t, J = 8.8 Hz, 2H); 19
F NMR (376 MHz,
CD3OD): –110.95 (d, JPF = 112 Hz), –117.32; 31
P NMR (162 MHz, CD3OD): 6.97 (t, JPF =
112 Hz). HRMS-ESI (m/z): [M+H]+ calcd. for C13H11F3O3P, 303.0392; found, 303.0387.
Inhibitor 3.31.
1H NMR (400 MHz, CD3OD): 7.73 (s, 1H), 7.62 (d, J = 7.7 Hz, 1H), 7.55
(d, J = 8.0 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H), 7.30 (d, J = 9.1 Hz, 1H),
7.08 (d, J = 8.2 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 3.79 (s, 3H); 19
F NMR (376 MHz, CD3OD):
−109.54 (d, JPF = 114 Hz); 31
P NMR (162 MHz, CD3OD): 7.18 (t, JPF = 114 Hz). HRMS-ESI
(m/z): [M−H]− calcd. for C14H12F2O4P, 313.0447; found, 313.0445.
Inhibitor 3.32.
1H NMR (400 MHz, CD3OD): 7.83–7.75 (m, 3H), 7.70 (d, J = 8.0 Hz, 1H),
7.62–7.48 (m, 4H); 19
F NMR (376 MHz, CD3OD): –64.07, –111.06 (d, JPF = 112 Hz); 31
P
NMR (162 MHz, CD3OD): 6.88 (t, JPF = 112 Hz). HRMS-ESI (m/z): [M+H]+ calcd. for
C14H11F5O3P, 353.0360; found, 353.0332.
Inhibitor 3.33.
1H NMR (400 MHz, CD3OD): 7.68 (s, 1H), 7.58 (d, J = 7.6 Hz, 1H), 7.53
(d, J = 7.9 Hz, 1H), 7.51–7.35 (m, 2H), 7.34–7.25 (m, 1H), 7.20–7.14 (m, 1H), 7.14–7.07 (m,
53
1H); 19
F NMR (376 MHz, CD3OD): –110.92 (d, JPF = 113 Hz), –120.12; 31
P NMR (162 MHz,
CD3OD): 7.25 (br). HRMS-ESI (m/z): [M+H]+ calcd. for C13H11F3O3P, 303.0392; found,
303.0384.
Inhibitor 3.34.
1H NMR (400 MHz, CD3OD): 8.04 (s, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.89
(s, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.72–7.60 (m, 3H); 19
F NMR (376
MHz, CD3OD): –111.04 (d, JPF = 112 Hz); 31
P NMR (162 MHz, CD3OD): 6.81 (t, JPF = 112
Hz). MS-ESI (m/z): [M−H]− calcd. for C14H9F2NO3P, 308.03; found, 308.0.
Inhibitor 3.35.
1H NMR (400 MHz, CD3OD): 8.08 (s, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.84
(d, J = 7.8 Hz, 1H), 7.78 (t, J = 7.7 Hz, 1H), 7.61 (t, J = 7.9 Hz, 1H), 7.44 (d, J = 7.5 Hz, 1H),
7.40 (t, J = 1.9 Hz, 1H), 7.18 (dd, J = 8.3, 2.5 Hz, 1H), 4.08 (s, 3H); 19
F NMR (376 MHz,
CD3OD): –110.82 (d, JPF = 113 Hz); 31
P NMR (162 MHz, CD3OD): 7.05 (t, JPF = 113 Hz).
HRMS-ESI (m/z): [M+H]+ calcd. for C14H14F2O4P, 313.0592; found, 313.0574.
Inhibitor 3.36.
1H NMR (400 MHz, CD3OD): 7.86 (s, 1H), 7.78 (d, J = 7.5 Hz, 1H), 7.70–
7.57 (m, 4H), 7.50 (d, J = 8.5 Hz, 2H); 19
F NMR (376 MHz, CD3OD): –110.97 (d, JPF = 112
Hz); 31
P NMR (162 MHz, CD3OD): 6.89 (t, JPF = 112 Hz). HRMS-ESI (m/z): [M+H]+ calcd.
for C13H11[35]
ClF2O3P, 319.0097; found, 319.0110.
54
Inhibitor 3.37.
1H NMR (400 MHz, CD3OD): 7.59–7.51 (m, 2H), 7.51–7.38 (m, 3H),
7.33–7.21 (m, 3H); 19
F NMR (376 MHz, CD3OD): –110.81 (d, JPF = 113 Hz); 31
P NMR (162
MHz, CD3OD): 6.97 (t, JPF = 113 Hz). HRMS-ESI (m/z): [M+H]+ calcd. for C13H11
[35]ClF2O3P,
319.0097; found, 319.0084.
Inhibitor 3.38.
1H NMR (400 MHz, CD3OD): 7.83 (s, 1H), 7.73 (d, J = 7.3 Hz, 1H),
7.60−7.48 (m, 4H), 7.33 (d, J = 8.3 Hz, 2H), 2.95 (hept, J = 6.9 Hz, 1H), 1.28 (d, J = 6.9 Hz,
6H); 19
F NMR (376 MHz, CD3OD): −109.67 (d, JPF = 113 Hz); 31
P NMR (162 MHz, CD3OD):
7.11 (br t). HRMS-ESI (m/z): [M–H]– calcd. for C16H16F2O3P, 325.0811; found, 325.0816.
Inhibitor 3.39.
1H NMR (400 MHz, CD3OD): 7.82 (s, 1H), 7.72 (d, J = 7.2 Hz, 1H),
7.60−7.49, (m, 4H), 7.28 (d, J = 8.0 Hz, 2H), 2.38 (s, 3H); 19
F NMR (376 MHz, CD3OD):
−109.69 (d, JPF = 113 Hz); 31
P NMR (162 MHz, CD3OD): 7.05 (t, JPF = 113 Hz). HRMS-ESI
(m/z): [M−H]− calcd. for C14H12F2O3P, 297.0498; found, 297.0500.
Inhibitor 3.40.
1H NMR (400 MHz, CD3OD): 7.85 (s, 1H), 7.75 (d, J = 7.5 Hz, 1H), 7.66–
7.50 (m, 2H), 7.37 (t, J = 7.9 Hz, 1H), 7.20 (dd, J = 7.8, 1.3 Hz, 1H), 7.16 (t, J = 2.1 Hz, 1H),
6.94 (dd, J = 8.2, 2.4 Hz, 1H), 4.69 (hept, J = 6.1 Hz, 1H), 1.36 (d, J = 6.0 Hz, 6H); 19
F NMR
55
(376 MHz, CD3OD): –110.87 (d, JPF = 113 Hz); 31
P NMR (162 MHz, CD3OD): 7.09 (t, JPF =
113 Hz). MS-ESI (m/z): [M−H]− calcd. for C16H16F2O4P, 341.08; found, 341.1.
Inhibitor 3.41.
1H NMR (400 MHz, CD3OD): 7.79 (s, 1H), 7.71 (d, J = 7.5 Hz, 1H), 7.54
(d, J = 7.6 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.27 (dd, J = 7.5, 1.7 Hz, 1H), 7.17 (td, J = 7.7, 1.6
Hz, 1H), 6.94−6.87 (m, 2H); 19
F NMR (376 MHz, CD3OD): −109.53 (d, JPF = 114 Hz); 31
P
NMR (162 MHz, CD3OD): 7.24 (t, JPF = 114 Hz). HRMS-ESI (m/z): [M−H]−
calcd. for
C13H10F2O4P, 299.0290; found, 299.0285.
Inhibitor 3.42.
1H NMR (400 MHz, CD3OD): 7.83 (s, 1H), 7.71 (d, J = 7.5 Hz, 1H), 7.58
(d, J = 8.0 Hz, 1H), 7.53 (t, J = 7.7 Hz, 1H), 7.45 (s, 1H), 7.41 (d, J = 7.7 Hz, 1H), 7.32 (t, J =
7.6 Hz, 1H), 7.18 (d, J = 7.5 Hz, 1H), 2.40 (s, 3H); 19
F NMR (376 MHz, CD3OD): −109.64 (d,
JPF = 113 Hz); 31
P NMR (162 MHz, CD3OD): 7.09 (t, JPF = 113 Hz). HRMS-ESI (m/z): [M–
H]– calcd. for C14H12F2O3P, 297.0498; found, 297.0498.
Inhibitor 3.43.
1H NMR (400 MHz, CD3OD): 7.83 (s, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.59
(d, J = 7.8 Hz, 1H), 7.54 (t, J = 7.7 Hz, 1H), 7.48 (s, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.37 (t, J =
7.6 Hz, 1H), 7.25 (d, J = 7.5 Hz, 1H), 2.97 (hept, J = 6.9 Hz, 1H), 1.29 (d, J = 7.0 Hz, 6H); 19
F
NMR (376 MHz, CD3OD): −109.67 (d, JPF = 113.4 Hz); 31
P NMR (162 MHz, CD3OD): 7.16
(br t). HRMS-ESI (m/z): [M−H]− calcd. for C16H16F2O3P, 325.0811; found, 325.0805.
56
Inhibitor 3.62.
1H NMR (400 MHz, DMSO-d6): 9.01 (s, 1H), 7.57, 7.53 (ABq, J = 8.0 Hz,
4H), 7.46−7.29 (m, 4H), 2.74 (s, 3H); 19
F NMR (376 MHz, DMSO-d6): −107.44 (d, JPF = 106
Hz); 31
P NMR (162 MHz, DMSO-d6): 2.83 (t, JPF = 106 Hz). HRMS-ESI (m/z): [M−H]− calcd.
for C14H13F2NO5PS, 376.0226; found, 376.0232.
Inhibitor 3.63.
1H NMR (400 MHz, DMSO-d6): 7.87−7.78 (m, 6H), 7.73 (d, J = 8.0 Hz,
2H); 19
F NMR (376 MHz, DMSO-d6): −111.49 (d, JPF = 112 Hz); 31
P NMR (162 MHz,
DMSO-d6): 5.61 (t, JPF = 112 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C14H9F2NO3P,
308.0294; found, 308.0297.
Inhibitor 3.64.
1H NMR (400 MHz, DMSO-d6): 7.70 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 8.0
Hz, 2H), 7.56 (d, J = 8.0 Hz, 2H), 7.41 (d, J = 8.0 Hz, 2H), 4.73 (q, J = 6.4 Hz, 1H), 1.32 (d, J =
6.4 Hz, 3H); 19
F NMR (376 MHz, DMSO-d6): −104.57 (d, JPF = 104 Hz); 31
P NMR (162 MHz,
DMSO-d6): 2.62 (t, JPF = 104 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C15H14F2O4P,
327.0603; found, 327.0608.
57
Inhibitor 3.65.
1H NMR (400 MHz, DMSO-d6): 8.04−7.96 (m, 2H), 7.78−7.71 (m, 5H),
7.67−7.63 (m, 1H); 19
F NMR (376 MHz, DMSO-d6): −111.49 (d, JPF = 112 Hz); 31
P NMR
(162 MHz, DMSO-d6): 5.81 (t, JPF = 112 Hz). HRMS-ESI (m/z): [M−H]− calcd. for
C14H9F2NO3P, 308.0294; found, 308.0296.
Inhibitor 3.66.
1H NMR (400 MHz, DMSO-d6): 7.87−7.85 (m, 1H), 7.84−7.74 (m, 3H),
7.67 (d, J = 8.0 Hz, 2H), 7.62−7.60 (m, 1H), 7.59−7.54 (m, 1H); 19
F NMR (376 MHz, DMSO-
d6): −111.15 (d, JPF = 111 Hz); 31
P NMR (162 MHz, DMSO-d6): 5.39 (t, JPF = 111 Hz).
HRMS-ESI (m/z): [M−H]− calcd. for C14H9F2NO3P, 308.0294; found, 308.0298.
Inhibitor 3.67.
1H NMR (400 MHz, DMSO-d6): 7.66, 7.63 (ABq, J = 8.8 Hz, 4H), 7.56 (d,
J = 8.8 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 4.65 (hept, J = 6.0 Hz, 1H), 1.33 (d, J = 6.0 Hz, 6H); 19
F NMR (376 MHz, DMSO-d6): −111.02 (d, JPF = 113 Hz); 31
P NMR (162 MHz, DMSO-d6):
5.93 (t, JPF = 113 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C16H16F2O4P, 341.0760; found,
341.0763.
58
Inhibitor 3.68.
1H NMR (400 MHz, DMSO-d6): 7.70−7.65 (m, 4H), 7.34 (t, J = 8.4 Hz,
1H), 7.19−7.13 (m, 2H), 6.92 (ddd, J = 8.4 Hz, 2.4, 0.8 Hz, 1H), 4.67 (hept, J = 6.0 Hz, 1H),
1.33 (d, J = 6.0 Hz, 6H); 19
F NMR (376 MHz, DMSO-d6): −111.15 (d, JPF = 113 Hz); 31
P NMR
(162 MHz, DMSO-d6): 5.88 (t, JPF = 113 Hz). HRMS-ESI (m/z): [M−H]− calcd. for
C16H16F2O4P, 341.0760; found, 341.0760.
Inhibitor 3.69.
1H NMR (400 MHz, DMSO-d6): 7.69 (d, J = 8.0 Hz, 2H), 7.65 (d, J = 8.0
Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 2.94 (hept, J = 6.4 Hz, 1H), 1.28 (d, J
= 6.4 Hz, 6H); 19
F NMR (376 MHz, DMSO-d6): −111.19 (d, JPF = 114 Hz); 31
P NMR (162
MHz, DMSO-d6): 5.99 (t, JPF = 114 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C16H16F2O3P,
325.0811; found, 325.0815.
Inhibitor 3.70.
1H NMR (400 MHz, DMSO-d6): 7.72−7.66 (m, 4H), 7.45−7.37 (m, 3H),
7.27−7.24 (m, 1H), 2.98 (hept, J = 6.4 Hz, 1H), 1.30 (d, J = 6.4 Hz, 6H); 19
F NMR (376 MHz,
DMSO-d6): −111.15 (d, JPF = 113 Hz); 31
P NMR (162 MHz, DMSO-d6): 5.95 (t, JPF = 113
Hz). HRMS-ESI (m/z): [M−H]− calcd. for C16H16F2O3P, 325.0811; found, 325.0811.
59
Inhibitor 3.71.
1H NMR (400 MHz, DMSO-d6): 9.86 (s, 1H), 7.70 (d, J = 8.0 Hz, 2H),
7.61 (d, J = 8.0 Hz, 2H), 7.47−7.39 (m, 3H), 7.25−7.23 (m, 1H), 3.03 (s, 3H); 19
F NMR (376
MHz, DMSO-d6): −108.08 (d, JPF = 107 Hz); 31
P NMR (162 MHz, DMSO-d6): 2.86 (t, JPF =
107 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C14H13F2NO5PS, 376.0226; found, 376.0230.
Inhibitor 3.72.
1H NMR (400 MHz, DMSO-d6): 7.68 (d, J = 8.0 Hz, 2H), 7.58 (d, J = 8.0
Hz, 2H), 7.34−7.27 (m, 2H), 7.22−7.18 (m, 2H), 7.12−7.05 (m, 4H), 6.82 (t, J = 7.2 Hz, 1H); 19
F
NMR (376 MHz, DMSO-d6): −108.01 (d, JPF = 107 Hz); 31
P NMR (162 MHz, DMSO-d6):
2.91 (t, JPF = 107 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C19H15F2NO3P, 374.0763; found,
374.0764.
Inhibitor 3.73.
1H NMR (400 MHz, DMSO-d6): 7.73 (d, J = 8.4 Hz, 2H), 7.65−7.56 (m,
3H), 7.53−7.48 (m, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.37−7.32 (m, 1H), 4.77 (q, J = 6.4 Hz, 1H),
1.34 (d, J = 6.4 Hz, 3H); 19
F NMR (376 MHz, DMSO-d6): −107.96 (d, JPF = 107 Hz); 31
P
NMR (162 MHz, DMSO-d6): 2.91 (t, JPF = 107 Hz). HRMS-ESI (m/z): [M−H]− calcd. for
C15H14F2O4P, 327.0603; found, 327.0602.
60
Inhibitor 3.74.
1H NMR (400 MHz, DMSO-d6): 7.32 (d, J = 8.0 Hz, 2H), 7.60−7.57 (m,
3H), 7.51 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H), 7.30−7.24 (m, 5H), 7.20−7.15 (m, 1H),
4.01 (s, 2H); 19
F NMR (376 MHz, DMSO-d6) −107.98 (d, JPF = 107 Hz); 31
P NMR (162 MHz,
DMSO-d6): 2.90 (t, JPF = 107 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C20H16F2O3P,
373.0811; found, 373.0803.
Synthesis of and analytical data for inhibitors 3.51–3.60
To synthesize inhibitors containing both an ortho hydroxyl and meta alkyl substituent
(general form 3.102), first the boronic acids had to be formed. This process required no
purifications starting from alkylphenols 3.44, so the sequence is written as one procedure
(Scheme 3.7). Compounds 3.51–3.56 and 3.58–3.59 were synthesized with this procedure.
Scheme 3.7. General synthesis of inhibitors 3.102
General synthesis of inhibitors 3.102. Selective ortho-bromination of alkylphenols was
accomplished via a modified literature procedure.19
A solution of N-bromosuccinimide (1.0
equiv) in CH2Cl2 (0.2 M) was added dropwise via cannula to a stirred solution of the phenol in
CH2Cl2 (0.5 M) containing catalytic N,N-diisopropylamine (0.1 equiv), the reaction flask having
been placed in an ambient temperature water bath. The reaction mixture was stirred for 90 min at
which point it was acidified with 1 N HCl to pH < 2.0. The reaction mixture was diluted with 1
volume of water. The layers were separated, and the organic layer was dried over MgSO4. The
volatile material was removed with a rotary evaporator under vacuum to afford the crude
products, generally as yellow to colorless liquids, which were typically used without further
purification.
Borylation of these phenols was accomplished following a literature procedure.20
n-
Butyllitium (2.5 M in hexanes, 2.15 equiv) was added dropwise via syringe to a stirred solution
of the starting o-bromophenol in Et2O (0.2 M) in a dry ice−acetone cold temperature bath. The
cold bath was removed and the reaction solution was allowed to warm to ambient temperature in
61
air for 2.5 h. The reaction flask was resuspended in the dry ice−acetone cold temperature bath.
After the mixture was stirred for 10 min in the cold bath, a solution of trimethyl borate (1.67
equiv, 1.5 M in Et2O) was added. After the mixture was stirred in the cold bath for 30 min, the
reaction mixture was stirred at ambient temperature for 18 h. The reaction mixture was quenched
with 0.5 volumes of 1 N HCl. The layers were separated, and the organic layer was dried over
MgSO4. The volatile material was removed with a rotary evaporator under vacuum to afford the
crude products as orange to brown viscous oils or sticky solids. Note: Although crude products
show multiple sets of peaks in the aromatic region when analyzed by 1H-NMR (potentially the
hydrolytic boroxine species), the crude products show only one major peak on LCMS which
corresponds to the mass of the boronic acids, and the products were used without further
purification.
Final inhibitor conversion was accomplished using general procedure A (vide supra).
Analytical data for each inhibitor is provided.
Inhibitor 3.51.
1H NMR (400 MHz, CD3OD): 7.72 (s, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.57
(d, J = 7.1 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.19 (dd, J = 7.8, 1.7 Hz, 1H), 7.04 (dd, J = 7.6, 1.7
Hz, 1H), 6.92 (t, J = 7.6 Hz, 1H), 3.38 (hept, J = 6.9 Hz, 1H), 1.26 (d, J = 6.9 Hz, 6H); 19
F NMR
(376 MHz, CD3OD): −109.81 (d, JPF = 114 Hz); 31
P NMR (162 MHz, CD3OD): 7.27 (t, JPF =
114 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C16H16F2O4P, 341.0760; found, 341.0755.
Inhibitor 3.52.
1H NMR (400 MHz, CD3OD): 7.78 (s, 1H), 7.70 (d, J = 7.5 Hz, 1H), 7.54
(d, J = 7.8 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.12 (d, J = 2.3 Hz, 1H), 7.06 (dd, J = 8.3, 2.3 Hz,
1H), 6.83 (d, J = 8.3 Hz, 1H), 2.87 (hept, J = 6.9 Hz, 1H), 1.24 (d, J = 6.9 Hz, 6H); 19
F NMR
(376 MHz, CD3OD): −109.48 (d, JPF = 114 Hz); 31
P NMR (162 MHz, CD3OD): 7.39 (t, JPF =
114 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C16H16F2O4P, 341.0760; found, 341.0756.
62
Inhibitor 3.53.
1H NMR (400 MHz, CD3OD): 7.73 (s, 1H), 7.63 (d, J = 7.4 Hz, 1H), 7.57
(d, J = 7.7 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.12 (dd, J = 7.5, 1.7 Hz, 1H), 7.06 (dd, J = 7.6, 1.7
Hz, 1H), 6.89 (t, J = 7.6 Hz, 1H), 2.70 (q, J = 7.5 Hz, 2H), 1.23 (t, J = 7.5 Hz, 3H); 19
F NMR
(376 MHz, CD3OD): −109.75 (d, JPF = 114 Hz); 31
P NMR (162 MHz, CD3OD): 7.25 (t, JPF =
114 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C15H14F2O4P, 327.0603; found, 327.0599.
Inhibitor 3.54.
1H NMR (400 MHz, CD3OD): 7.68 (s, 1H), 7.63−7.50 (m, 3H), 7.26 (d, J =
7.7 Hz, 1H), 7.02 (d, J = 7.5 Hz, 1H), 6.87 (t, J = 7.7 Hz, 1H), 1.44 (s, 9H); 19
F NMR (376 MHz,
CD3OD): −110.00 (d, JPF = 113 Hz); 31
P NMR (162 MHz, CD3OD): 6.96 (t, JPF = 113 Hz).
HRMS-ESI (m/z): [M−H]− calcd. for C17H18F2O4P, 355.0916; found, 355.0913.
Inhibitor 3.55.
1H NMR (400 MHz, CD3OD): 7.63 (s, 1H), 7.53 (d, J = 7.4 Hz, 1H), 7.50–
7.37 (m, 2H), 7.00 (d, J = 7.4 Hz, 1H), 6.95 (d, J = 7.6 Hz, 1H), 6.77 (t, J = 7.5 Hz, 1H), 2.59–
2.52 (m, 2H), 1.55 (sext, J = 7.4 Hz, 2H), 0.88 (t, J = 7.4 Hz, 3H); 19
F NMR (376 MHz,
CD3OD): –110.89 (d, JPF = 113 Hz); 31
P NMR (162 MHz, CD3OD): 7.24 (br). HRMS-ESI
(m/z): [M−H]− calcd. for C16H16F2O4P, 341.0760; found, 341.0766.
63
Inhibitor 3.56.
1H NMR (400 MHz, CD3OD): 7.62 (s, 1H), 7.56–7.37 (m, 3H), 7.05 (d, J =
7.5 Hz, 1H), 6.94 (d, J = 7.5 Hz, 1H), 6.83 (t, J = 7.5 Hz, 1H), 3.12–3.01 (m, 1H), 1.65–1.45 (m,
2H), 1.13 (d, J = 6.9 Hz, 3H), 0.78 (t, J = 7.3 Hz, 3H); 19
F NMR (376 MHz, CD3OD): –110.94
(d, JPF = 113 Hz); 31
P NMR (162 MHz, CD3OD): 7.24 (t, JPF = 113 Hz). HRMS-ESI (m/z):
[M−H]− calcd. for C17H18F2O4P, 355.0916; found, 355.0907.
Inhibitor 3.58.
1H NMR (400 MHz, CD3OD): 7.72 (s, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.57
(d, J = 7.7 Hz, 1H), 7.52 (t, J = 7.7 Hz, 1H), 7.20 (dd, J = 7.6, 1.7 Hz, 1H), 7.03 (dd, J = 7.6, 1.7
Hz, 1H), 6.91 (t, J = 7.6 Hz, 1H), 3.46−3.33 (m, 1H), 2.12−2.01 (m, 2H), 1.92−1.78 (m, 2H),
1.78−1.56 (m, 4H); 19
F NMR (376 MHz, CD3OD): −109.57 (d, JPF = 112 Hz); 31
P NMR (162
MHz, CD3OD) 7.19 (br t). HRMS-ESI (m/z): [M−H]− calcd. for C18H18F2O4P, 367.0916;
found, 367.0912.
Inhibitor 3.59.
1H NMR (400 MHz, CD3OD): 7.72 (s, 1H), 7.61 (d, J = 7.3 Hz, 1H), 7.57
(d, J = 7.6 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.17 (dd, J = 7.6, 1.7 Hz, 1H), 7.03 (dd, J = 7.6, 1.7
Hz, 1H), 6.91 (t, J = 7.6 Hz, 1H), 3.08−2.95 (m, 1H), 1.93−1.82 (m, 4H), 1.82−1.73 (m, 1H),
1.56−1.23 (m, 5H); 19
F NMR (376 MHz, CD3OD): −109.61 (d, JPF = 113 Hz); 31
P NMR (162
MHz, CD3OD): 7.29 (t, JPF = 113 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C19H20F2O4P,
381.1073; found, 381.1068.
64
Inhibitors 3.57 and 3.60 could not be made by the above method, so were made by a different
sequence.
Compound 3.103. Following a previous report, dilithiation of 2-bromophenol was
accomplished.20
2-Bromophenol (3.5 g, 20 mmol, 1.0 equiv) was dissolved in Et2O (55 mL) and
cooled in a dry ice−acetone cold bath. After the mixture was stirred for 10 min, n-butyllithium
(1.6 M in hexanes, 26.5 mL, 42 mmol, 2.1 equiv) was added dropwise by syringe. The cold bath
was removed and the reaction mixture was stirred at ambient temperature for 2 h, resulting in the
dilithiated reagent. Note: LCMS of an acid quenched aliquot confirmed that there was no starting
bromide remaining. An amount of 19 mL (approximately 5 mmol) of the above reagent was
transferred to a reaction flask charged with a stir bar and was suspended in the dry ice−acetone
cold bath. Next, 392 L of cyclobutanone (5.5 mmol, 1.1 equiv) was added neat via syringe. The
reaction mixture was allowed to warm to ambient temperature and was stirred for 14 h. The
reaction was quenched by addition of 10 mL of saturated ammonium chloride. The layers were
separated, and the water layer was washed with 15 mL of Et2O. The combined organic layers
were dried over MgSO4, and the volatile material was removed with a rotary evaporator under
vacuum to afford the crude benzyl alcohol product as a yellow liquid (880 mg). The crude
product (~5 mmol) was dissolved in 5 mL of CH2Cl2 in a 20 mL scintillation vial open to the
atmosphere, and triethylsilane (2.4 mL, 15 mmol, ~3 equiv) was added followed by 2.5 mL of
trifluoroacetic acid (1:0.5 by volume). The reaction mixture was stirred at ambient temperature
for 16 h. The volatile material was removed with a rotary evaporator under vacuum to afford the
crude product. The crude product was purified via gradient column chromatography (2−20%
ethyl acetate in hexanes) to afford the product as a clear oil (550 mg, 67%). 1H NMR (400 MHz,
CDCl3): 7.18 (dt, J = 7.6, 1.3 Hz, 1H), 7.09 (td, J = 7.7, 1.7 Hz, 1H), 6.92 (td, J = 7.4, 1.1 Hz,
1H), 6.76 (dd, J = 7.9, 1.2 Hz, 1H), 4.63 (s, 1H), 3.75−3.58 (m, 1H), 2.46−2.30 (m, 2H),
2.26−2.12 (m, 2H), 2.12−2.00 (m, 1H), 1.95−1.80 (m, 1H). MS-ESI (m/z): [M+H]+
calcd. for
C10H13O, 148.10; found, 149.1.
Compound 3.104. Borylation of 2-cyclobutylphenol (3.103) was accomplished by closely
following a previous report.21
A 1 dram oven-dried vial was charged with a stir bar and brought
into an inert atmosphere glovebox where it was charged with 3.103 (222 mg, 1.5 mmol, 1.0
equiv), diethylsilane (290 L, 2.25 mmol, 1.5 equiv), bis(1,5-cyclooctadiene)diiridium(I)
dichloride (10 mg, 0.015 mmol, 0.01 equiv, 2 mol % Ir), and 3 mL of benzene (0.5 M). The vial
was capped, removed from the inert atmosphere glovebox, and the reaction mixture was stirred
at ambient temperature for 1 h. Solvent was removed with a rotary evaporator under vacuum,
and the reaction vial was brought back into the inert atmosphere glovebox where it was charged
65
with bis(pinacolato)diboron (381 mg, 1.5 mmol, 1.0 equiv), pinacolborane (21.8 L, 0.15 mmol,
10 mol %), bis(1,5-cyclooctadiene)diiridium(I) dichloride (10 mg, 0.015 mmol, 0.01 equiv, 2
mol % Ir), 4,4-di-tert-butylbipyridine (8 mg, 0.03 mmol, 2 mol %), and 3 mL of THF (0.5 M).
The vial was capped, removed from the inert atmosphere glovebox, and placed in a heating block
preheated to 80 °C, and the mixture was stirred vigorously for 2 h. The vial was cooled to
ambient temperature, and saturated potassium hydrogen difluoride (~4.5 M, 3 mL, ~13.5 mmol,
9 equiv) was added. The resulting reaction mixture was stirred vigorously for 18 h. The volatile
material was removed with a rotary evaporator under vacuum to afford the crude product. The
crude product was dissolved in 5 mL of hot acetone, and the insoluble salts were filtered. The
volatile components of the filtrate were removed with a rotary evaporator under vacuum until 1
mL of acetone remained. The solution was transferred to a 15 mL conical centrifuge tube, and
approximately 5 mL of pentane was added to the acetone solution to precipitate the product. The
suspension was centrifuged to pellet the solid product, and the black liquid was removed.
Additional washes were accomplished by adding 5 mL of pentane, resuspending the solid
followed by centrifugation to pellet the solid products for a total of three additional washes.
Removal of residual solvent under vacuum afforded the product as a light gray solid (200 mg,
52%). 1H NMR (400 MHz, acetone-d6): 7.62 (q, J = 11.8 Hz, 1H), 7.15 (d, J = 7.0 Hz, 1H),
6.90 (dd, J = 7.5, 1.8 Hz, 1H), 6.60 (t, J = 7.3 Hz, 1H), 3.74 (p, J = 8.5 Hz, 1H), 2.33−2.18 (m,
2H), 2.15−2.03 (m, 1H), 2.03−1.86 (m, 2H), 1.84−1.71 (m, 1H). MS-ESI (m/z): [M(boronic acid)−H]
– calcd. for C10H12BO3, 191.09; found 191.1.
Inhibitor 3.57. For the Suzuki-Miyaura cross-coupling, general procedure B was followed
with 90 mg (0.35 mmol) of the potassium trifluoroborate 3.104 and 100 mg (0.35 mmol) of
arylbromide 3.21. The procedure yielded 18 mg (15%) of the desired compound as a white
hygroscopic powder. 1H NMR (400 MHz, CD3OD): 7.71 (s, 1H), 7.61−7.54 (m, 2H), 7.49 (t, J
= 7.7 Hz, 1H), 7.20 (dd, J = 7.4, 1.6 Hz, 1H), 7.05 (dd, J = 7.6, 1.7 Hz, 1H), 6.92 (t, J = 7.5 Hz,
1H), 3.82 (p, J = 8.6 Hz, 1H), 2.43−2.32 (m, 2H), 2.23−1.96 (m, 3H), 1.93−1.75 (m, 1H); 19
F
NMR (376 MHz, CD3OD): −109.20 (d, JPF = 109 Hz); 31
P NMR (162 MHz, CD3OD): 6.97
(br t). HRMS-ESI (m/z): [M−H]– calcd. for C17H16F2O4P, 353.0760; found 353.0754.
66
Inhibitor 3.60. Inhibitor 3.60 was synthesized by the same procedures described for 3.57,
yielding the product in a 1:0.4 ratio with residual trifluoroacetic acid (as determined by 19
F
NMR). 1H NMR (400 MHz, CD3OD): 7.72 (s, 1H), 7.68−7.44 (m, 3H), 7.13 (dd, J = 7.6, 1.7
Hz, 1H), 6.99 (dd, J = 7.4, 1.7 Hz, 1H), 6.87 (t, J = 7.6 Hz, 1H), 3.29−3.16 (m, 1H), 2.00−1.49
(m, 12H); 19
F NMR (376 MHz, CD3OD): −109.61 (d, JPF = 112 Hz); 31
P NMR (162 MHz,
CD3OD): 7.01 (br t). HRMS-ESI (m/z): [M−H]– calcd. for C20H22F2O4P, 395.1229; found
395.1225.
Synthesis of and analytical data for inhibitors 3.79–3.83 and 3.61
Compound 3.105. To a solution of 1-bromo-3-iodobenzene 3.17 (2.0 g, 7.1 mmol) in THF
(15 mL) at –78°C under N2 atmosphere was added dropwise n-BuLi (2.5 M in hexanes, 2.8 mL,
7.0 mmol). The reaction mixture was stirred at the same temperature for 0.5 h. 3,4-
Dichlorobenzaldehyde (1.36 g, 7.77 mmol) was added and the reaction mixture was kept at –78
°C for 2 h. Saturated NH4Cl (50 mL) and Et2O (50 mL) were added, and the reaction mixture
was allowed to warm to ambient temperature. The phases were separated and the aqueous phase
was extracted with Et2O (100 mL). The combined organic phase was dried over MgSO4, filtered
and concentrated to yield 3.105 as a viscous oil (1.85 g, 72%). 1H NMR (400 MHz, CDCl3): δ
7.51 (m, 1H), 7.48 (m, 1H), 7.44−7.40 (m, 2H), 7.27−7.16 (m, 3H), 5.74 (d, J = 3.2 Hz, 1H),
2.31 (d, J = 3.2 Hz, 1H).
Compound 3.106. Compound 3.105 (1.80 g, 5.42 mmol) was dissolved in dichloromethane
(6 mL). Et3SiH (3.0 mL, 18.7 mmol) was added at ambient temperature, followed by
trifluoroacetic acid (TFA, 3.0 mL). The resulting solution was stirred at ambient temperature for
15 h. Volatiles were removed under reduced pressure. The residue was purified by automated
silica gel flash column chromatography (80 g flash column, column volume = 125 mL, 60
mL/min, linear gradient of 0−20% Et2O in hexanes over 25 min) to yield 3.106 as a white solid (1.57 g, 92% yield).
1H NMR (400 MHz, CDCl3): δ 7.38−7.35 (m, 2H), 7.31−7.30 (m, 1H), 7.25
67
(d, J = 2.0 Hz, 1H), 7.17 (t, J = 8.0 Hz, 1H), 7.09−7.06 (m, 1H), 7.00 (dd, J = 8.0, 2.0 Hz, 1H),
3.89 (s, 2H).
Compound 3.85. . To a Schlenk flask equipped with a stirring bar was added 3.106 (1.70 g,
5.38 mmol), bis(pinacolato)diboron (B2pin2, 2.73 g, 10.8 mmol), KOAc (2.12 g, 21.6 mmol), and
DMSO (22 mL). Nitrogen was bubbled through the reaction mixture for 0.5 h. Pd(dppf)Cl2 (0.22
g, 0.27 mmol) was added. Nitrogen was bubbled through the reaction mixture for an additional
0.5 h. The reaction mixture was then stirred at 80 °C for 6 h, cooled to ambient temperature, and
poured into water (220 mL). The resulting suspension was extracted with Et2O (2 × 150 mL).
The combined organic extracts was dried over anhydrous MgSO4, concentrated, and the residue
was purified by automated silica gel flash column chromatography (80 g flash column, column
volume = 125 mL, 60 mL/min, linear gradient of 0−20% Et2O in hexanes over 25 min) to yield
3.85 as a white solid (1.60 g, 82% yield). 1H NMR (400 MHz, CDCl3): δ 7.69 (d, J = 8.0 Hz,
1H), 7.64 (s, 1H), 7.32 (t, J = 8.0 Hz, 2H), 7.26−7.22 (m, 2H), 7.0 (d, J = 8.0 Hz, 1H), 3.92 (s,
2H), 1.35 (s, 12H).
Inhibitor 3.81. Compound 3.81 was prepared using general Suzuki-Miyaura cross-coupling
procedure B starting with 80 mg (0.28 mmol) of 3.22 and 152 mg (0.42 mmol) of 3.85. The procedure yielded 34 mg (28%) of 3.81 as a white solid.
1H NMR (400 MHz, DMSO-d6): δ 7.74
(d, J = 8.0 Hz, 2H), 7.62−7.58 (m, 4H), 7.54−7.51 (m, 2H), 7.40 (t, J = 8.0 Hz, 1H), 7.25−7.29 (m, 2H), 4.01 (s, 2H);
19F NMR (376 MHz, DMSO-d6): δ −107.62 (d, JPF = 104 Hz);
31P NMR
(162 MHz, DMSO-d6): δ 2.66 (t, JPF = 104 Hz). HRMS-ESI (m/z): [M−H]− calcd. for
C20H14F2[35]
Cl2O3P, 441.0031; found, 441.0030.
Inhibitors 3.79 and 3.80 were synthesized using the same procedures as 3.81. Analytical data
for each inhibitor is provided.
68
Inhibitor 3.79.
1H NMR (400 MHz, DMSO-d6): 7.72 (d, J = 8.0 Hz, 2H), 7.60−7.51 (m,
3H), 7.51 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H), 7.34−7.29 (m, 2H), 7.24 (d, J = 8.0 Hz,
1H), 7.12−7.06 (m, 2H), 4.00 (s, 2H); 19
F NMR (376 MHz, DMSO-d6): −107.78 (d, JPF = 107
Hz), −117.29; 31
P NMR (162 MHz, DMSO-d6): 2.90 (t, JPF = 107 Hz). HRMS-ESI (m/z):
[M−H]− calcd. for C20H15F3O3P, 391.0716; found, 391.0719.
Inhibitor 3.80.
1H NMR (400 MHz, DMSO-d6): 7.69 (d, J = 8.0 Hz, 2H), 7.58−7.56 (m,
3H), 7.53−7.51 (m, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.33−7.28 (m, 4H), 7.22 (d, J = 7.6 Hz, 1H),
3.99 (s, 2H); 19
F NMR (376 MHz, DMSO-d6): −108.01 (d, JPF = 104 Hz); 31
P NMR (162 MHz,
DMSO-d6): 2.59 (t, JPF = 104 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C20H15F2
[35]ClO3P,
407.0421; found, 407.0418.
Compound 3.108. To a solution of known compound 3.107
32 (1.5 g, 5.3 mmol) in THF (45
mL) was added n-BuLi (2.5 M in hexanes, 2.1 mL, 5.3 mmol) dropwise at −78 °C under N2
atmosphere. The resulting solution was stirred at the same temperature for 0.5 h. 3,4-
Dichlorobenzaldehyde (0.93 g, 5.3 mmol in THF (3 mL) was added and the reaction mixture was
stirred at −78 °C for 1 h. 1 N HCl (16.5 mL) and Et2O (100 mL) were added. The reaction
mixture was allowed to warm to ambient temperature. The layers were separated, and the
aqueous layer was extracted with Et2O (2 × 100 mL). The combined organic solution was dried
over anhydrous MgSO4 and concentrated under reduced pressure to give 3.108, which was used
in the following step without purification.
69
Inhibitor 3.82. Inhibitor 3.82 was prepared using general Suzuki-Miyaura cross-coupling
procedure B using 80 mg (0.28 mmol) of 3.22 and 124 mg (0.42 mmol) of 3.108. Inhibitor 3.82
was obtained in 36% yield (46 mg) as a white solid. 1H NMR (400 MHz, DMSO-d6): 7.74 (d, J
= 8.0 Hz, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.65 (s, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 8.0
Hz, 1H), 7.53 (d, J = 2.0 Hz, 1H), 7.46 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.42 (t, J = 8.0 Hz, 1H),
7.31 (d, J = 8.0 Hz, 1H), 6.04 (s, 1H); 19
F NMR (376 MHz, DMSO-d6): −108.09 (d, JPF = 107
Hz); 31
P NMR (162 MHz, DMSO-d6): 2.91 (t, JPF = 107 Hz). HRMS-ESI (m/z): [M−H]− calcd.
for C20H14[35]
Cl2F2O4P, 456.9980; found, 456.9979.
Compound 3.86. Compound 3.86 was prepared using general Suzuki-Miyaura cross-
coupling procedure B using 80 mg (0.28 mmol) of compound 3.8423
and 152 mg (0.42 mmol) of
3.85, yielding in 49 mg (42%) as a white solid. 1H NMR (400 MHz, DMSO-d6): 8.26 (d, J =
8.0 Hz, 2H), 7.84 (d, J = 8.0 Hz, 2H), 7.70 (s, 1H), 7.60−7.58 (m, 2H), 7.52 (d, J = 8.0 Hz, 1H),
7.43 (t, J = 8.0 Hz, 1H), 7.32−7.28 (m, 2H), 4.03 (s, 2H); 31
P NMR (162 MHz, DMSO-d6):
−3.07. HRMS-ESI (m/z): [M−H]− calcd. for C20H14
[35]Cl2O4P, 419.0012; found, 419.0020.
Inhibitor 3.83. To a solution of 3.86 (35 mg, 0.083 mmol) in MeOH (0.2 mL) at 0 °C was
added NaBH4 (16 mg, 0.41 mmol). The reaction mixture was stirred at 0 °C for 20 min. More
NaBH4 (16 mg) was added, and the reaction mixture was stirred for an additional 20 min and
acetic acid (0.1 mL) was then added. The reaction mixture was purified by automated reversed-
phase column chromatography (liquid injection, 50 g C18 column, column volume = 42.6 mL,
40 mL/min, linear gradient of 5−100% acetonitrile/water with 0.1% trifluoroacetic acid over 20
70
min) to yield 3.83 as a white solid (20 mg, 57% yield). 1H NMR (400 MHz, DMSO-d6):
7.58−7.45 (m, 8H), 7.36 (t, J = 7.6 Hz, 1H), 7.28 (dd, J = 7.6, 2.0 Hz, 1H), 7.20 (d, J = 7.6 Hz,
1H), 4.70 (d, J = 14.0 Hz, 1H), 4.00 (s, 2H); 31
P NMR (162 MHz, DMSO-d6): 18.03. HRMS-
ESI (m/z): [M−H]− calcd. for C20H16
[35]Cl2O4P, 421.0169; found, 421.0155.
Inhibitor 3.61 was synthesized using the same procedures as 3.83 beginning from known
monosodium (3-bromobenzoyl)phosphonate.23
Analytical data. 1H NMR (400 MHz, CD3OD):
7.51 (s, 1H), 7.40 (d, J = 7.3 Hz, 1H), 7.35−7.26 (m, 2H), 7.04 (dd, J = 7.6, 1.8 Hz, 1H), 6.91
(dd, J = 7.6, 1.7 Hz, 1H), 6.78 (t, J = 7.5 Hz, 1H), 4.84 (d, J = 13.2 Hz, 1H), 3.01−2.86 (m, 1H),
1.86−1.73 (m, 4H), 1.72−1.63 (m, 1H), 1.44−1.55 (m, 5H); 31
P NMR (162 MHz, CD3OD):
22.45. HRMS-ESI (m/z): [M−H]– calcd. for C19H22O5P, 361.1210; found, 361.1215.
Synthesis of and analytical data for diastereomeric inhibitors 3.97–3.100 and inhibitor 3.101
Compounds 3.88 and 3.89. Tris-[(1S,2R,5S)-menth-2-yl]phosphite is prepared according to
literature procedure.24
To a solution of phosphorus trichloride (1.7 mL, 20.0 mmol) in anhydrous
toluene (30 mL) at −20 °C was added slowly a solution of D-menthol (9.4 g, 60.0 mmol) and
triethylamine (10 mL) in toluene (50 mL). Upon complete addition, the cooling bath was
removed and the reaction mixture was stirred at ambient temperature for 5 h. The white
precipitate was filtered off and the filtrate was concentrated under reduced pressure to give tris-
[(1S,2R,5S)-menth-2-yl]phosphite (9.2 g), which was then treated with 4-bromobenzaldehyde
(3.4 g, 18.4 mmol) and chlorotrimethylsilane (5.4 mL, 42.6 mmol) at 0 °C. The suspension was
stirred at 0 °C for 2 h and then at ambient temperature for an additional 1 h. Volatiles were
removed under reduced pressure. The residue was purified by automatic silica gel column
chromatography (220 g flash column, column volume = 334 mL, 150 mL/min, linear gradient of
0−10% hexane/ethyl acetate over 35 min) to give the product as a mixture of diastereoisomers in
a ratio of 1.0:0.7 (8.8 g, 88%). The diastereoisomeric mixture was dissolved in acetonitrile (1
g/100 mL) at 60 °C and cooled to ambient temperature. The solid was collected by filtration and
recrystallized in acetonitrile one more time to give diastereomerically pure 3.89. The filtrate
solution was cooled to 0 °C. The solid was collected and recrystallized in acetonitrile one more
time to give diastereomerically pure 3.88. Compound 3.88. 1H NMR (400 MHz, CDCl3): 7.45
71
(d, J = 8.0 Hz, 2H), 7.37 (dd, J = 8.0, 4.0 Hz, 2H), 4.92 (dd, J = 11.2, 5.2 Hz, 1H), 4.24−4.15 (m,
2H), 3.42 (br, 1H), 2.19−2.14 (m, 1H), 2.06−1.97 (m, 2H), 1.81−1.77 (m, 1H), 1.65−1.59 (m,
4H), 1.41−0.85 (m, 19 H), 0.80 (d, J = 7.2 Hz, 3H), 0.76 (d, J = 6.8 Hz, 3H), 0.69 (d, J = 7.2 Hz,
3H); 31
P NMR (162 MHz, CDCl3): 18.75. Compound 3.89. 1H NMR (400 MHz, CDCl3):
7.45 (d, J = 8.0 Hz, 2H), 7.35 (dd, J = 8.0, 4.0 Hz, 2H), 4.91 (dd, J = 11.2, 5.2 Hz, 1H),
4.26−4.16 (m, 2H), 3.36 (dd, J = 9.2, 4.8 Hz, 1H), 2.16−2.13 (m, 1H), 1.99−1.91 (m, 3H),
1.70−1.59 (m, 4H), 1.42−0.79 (m, 22H), 0.76 (d, J = 7.2 Hz, 3H), 0.72 (d, J = 6.8 Hz, 3H); 31
P
NMR (162 MHz, CDCl3): 19.06.
Compound 3.91. To a suspension of 3.89 (1.8 g, 3.3 mmol) and NaI (2.0 g, 13.3 mmol) in
acetonitrile (20 mL) under nitrogen atmosphere was added chlorotrimethylsilane (1.7 mL, 13.5
mmol). The reaction mixture was refluxed for 10 h and then cooled to ambient temperature. The
precipitates were filtered off. The filtrate was concentrated under reduced pressure. The residue
was purified by automated reversed-phase column chromatography (150 g C18 column, column
volume = 130 mL, 85 mL/min, liquid injection, linear gradient of 5−30% acetonitrile in water
with 0.1% trifluoroacetic acid over 25 min) to give the title compound as a white solid (0.52 g,
59%). 1H NMR (400 MHz, DMSO-d6): 7.48 (d, J = 8.0 Hz, 2H), 7.33 (dd, J = 8.0, 4.0 Hz, 2H),
4.62 (d, J = 12.0 Hz, 1H); 31
P NMR (162 MHz, DMSO-d6): 17.43.
Compound 3.109. A mixture of 3.91 (43 mg, 0.16 mmol) and 10% Pd/C (dry, 16 mg) in
MeOH (4 mL) was stirred under H2 atmosphere (1 atm) for 3 h. The reaction mixture was
filtered through a syringe filter (0.2 m). The filtrate was concentrated to give the title compound
(28 mg, 93%). 1H NMR (400 MHz, DMSO-d6): 7.39−7.17 (m, 5H), 4.64 (d, J = 14.0 Hz, 1H);
31P NMR (162 MHz, DMSO-d6): 18.14.
Compound 3.91 was dissolved in ethanol (0.5 mL), and an excess of cyclohexylamine (100
L, 0.87 mmol) was added. The white precipitate was collected by filtration. []20
D : −10.6 (c
0.77, 50% MeOH/H2O at 20 °C) (lit.25a
−13.8, c 0.77, 50% MeOH/H2O).
72
Compound 3.90. Compound 3.90 (0.22 g) was obtained as a white solid in 45% yield
starting from 3.88 (1.0 g, 1.8 mmol) following the procedure described for the synthesis of
compound 3.91. The 1H NMR and
31P NMR spectra are the same as those of 3.91.
Compound 3.93. Compound 3.93 was prepared by adapting a literature procedure.
26a To a
solution of 3-bromo-1,2-dichlorobenzene (1.00 g, 4.42 mmol) in tert-butyl methyl ether (5.50
mL) at −78 °C was added dropwise n-BuLi (2.5 M in hexanes, 1.77, 4.42 mmol). The reaction
mixture was stirred at −78 °C for 1 h. The dry ice−acetone cooling bath was replaced with an
ice−water cooling bath. Anhydrous ZnCl2 (0.63 g, 4.64 mmol) was added. The resulting
suspension was stirred at 0 °C for 1 h. Additional n-BuLi (2.5 M in hexanes, 1.77 mL, 4.42
mmol) was added. The cooling bath was removed, and the reaction mixture was stirred at
ambient temperature for 4.5 h. Toluene (anhydrous, 22.5 mL) and tetraethylethylenediamine
(0.37 mL, 1.77 mmol) were added. The reaction mixture was stirred at ambient temperature for
an additional 1 h. (+)-3-exo-(Morpholino)isoborneol ((+)-MIB,26
27 mg, 0.11 mmol) was added.
and the reaction mixture was cooled to 0 °C and stirred for 0.5 h. 3-Bromobenzaldehyde (0.78 g,
2.21 mmol) was added, and the reaction mixture was stirred at 0 °C for 17 h. Water (30 mL) and
Et2O (30 mL) were added. The phases were separated, and the aqueous layer was extracted with
Et2O (2 × 50 mL). The combined organic phase was dried over anhydrous MgSO4, filtered, and
concentrated. The residue was purified by automated silica gel column chromatography (80 g
flash column, column volume = 125 mL, 60 mL/min, linear gradient of 0−50% ethyl acetate in
hexane over 25 min) to afford 3.93 as a colorless oil (0.51 g, 70%). The enantiomeric excess was
determined by HPLC (AS-H column, isopropanol/hexane = 5:95, flow rate = 0.5 mL/min, tr,major
= 32.3 min, minor enantiomer tr,minor = 35.4 min, 90% ee). 1H NMR (400 MHz, CDCl3): 7.52
(s, 1H), 7.48 (d, J = 2.0 Hz, 1H), 7.44−7.40 (m, 2H), 7.27−7.20 (m, 2H), 7.18 (dd, J = 8.4. 2.0
Hz, 1H), 5.75 (d, J = 3.2 Hz, 1H), 2.30 (d, J = 3.2 Hz, 1H).
Compound 3.94. Compound 3.94 (0.49 g) was obtained as a colorless oil in 67% yield
starting from 3-bromo-1,2-dichlorobenzene (1.00 g, 4.42 mmol) by following the procedure used
for the synthesis of 3.93 but replacing the catalyst (+)-MIB with (−)-MIB. The enantiomeric
excess was determined by HPLC (AS-H column, isopropanol/hexane = 5:95, flow rate = 0.5
73
mL/min, tr,major = 35.4 min, tr,minor = 32.3 min, 90% ee). The 1H NMR spectrum is the same as
that of 3.93.
Compound 3.95. Compound 3.95 (0.28 g) was obtained as a colorless oil in 64% yield
starting from 3.93 (0.38 g, 1.14 mmol) by following the procedure described for the synthesis of
3.85. 1H NMR (400 MHz, CDCl3): 7.80−7.79 (m, 1H), 7.75 (dt, J = 6.8, 1.2 Hz, 1H), 7.51 (dd,
J = 2.4, 0.8 Hz, 1H), 7.44−7.33 (m, 3H), 7.20 (ddd, J = 8.4, 2.4, 0.8 Hz, 1H), 5.80 (d, J = 3.2 Hz,
1H), 2.23 (d, J = 3.2 Hz, 1H), 1.35 (s, 12H).
Compound 3.96. Compound 3.96 was prepared in 58% yield starting from 3.94 (0.50 g, 1.51
mmol) by following the procedure described the synthesis of 3.85. The 1H NMR spectrum of
3.96 is the same as that of 3.95.
Inhibitors 3.97–3.100 were synthesized using the general Suzuki-Miyaura cross-coupling
procedure B beginning with either aryl bromide 3.90 or 3.91 and either aryl boronic ester 3.95 or
3.96. Inhibitor 3.101 was synthesized using the general Suzuki-Miyaura cross-coupling
procedure B beginning with commercially available 4-bromobenzylphosphonic acid and boronic
acid 3.108. Analytical data for each inhibitor is provided.
Inhibitor 3.97.
1H NMR (400 MHz, DMSO-d6): 7.69−7.67 (m, 2H), 7.56−7.54 (m, 3H),
7.51−7.45 (m, 3H), 7.41−7.36 (m, 2H), 7.34 (d, J = 8.0 Hz, 1H), 6.17 (s, 1H), 5.79 (s, 1H), 4.70
(d, J = 14.0 Hz, 1H); 31
P NMR (162 MHz, DMSO-d6): 17.97. HRMS-ESI (m/z): [M−H]− calcd.
for C20H16[35]
Cl2O5P, 437.0118; found, 437.0110.
74
Inhibitor 3.98.
1H NMR (400 MHz, DMSO-d6): 7.69−7.67 (m, 2H), 7.56−7.54 (m, 3H),
7.51−7.45 (m, 3H), 7.41−7.36 (m, 2H), 7.34 (d, J = 8.0 Hz, 1H), 6.17 (s, 1H), 5.79 (s, 1H), 4.70
(d, J = 14.0 Hz, 1H); 31
P NMR (162 MHz, DMSO-d6): 18.00. HRMS-ESI (m/z): [M−H]− calcd.
for C20H16[35]
Cl2O5P, 437.0118; found, 437.0111.
Inhibitor 3.99. The
1H NMR and
31P NMR spectra are the same as those reported for the
enantiomer, 3.98. HRMS-ESI (m/z): [M−H]− calcd. for C20H16
[35]Cl2O5P, 437.0118; found,
437.0111.
Inhibitor 3.100. The
1H NMR and
31P NMR spectra are the same as those reported for the
enantiomer, 3.97. HRMS-ESI (m/z): [M−H]− calcd. for C20H16
[35]Cl2O5P, 437.0118; found,
437.0112.
75
Inhibitor 3.101.
1H NMR (400 MHz, DMSO-d6): 7.65 (d, J = 4.0 Hz, 2H), 7.55−7.50 (m,
3H), 7.48 (d, J = 8.0 Hz, 1H), 7.39−7.35 (m, 2H), 7.32−7.30 (m, 3H), 6.15 (br, 1H), 5.78 (s, 1H),
2.97 (d, JHP = 21.2 Hz, 2H); 31
P NMR (162 MHz, DMSO-d6): 20.79. HRMS-ESI (m/z):
[M−H]− calcd. for C20H16
[35]Cl2O4P, 421.0169; found, 421.0162.
Assay procedures
Determination of substrate Km
To facilitate screening in a high-throughput fashion, 96-well plates were used with reaction
volumes of 100 L. An amount of 30 L of water was added to each well, followed by 5 L of
20× buffer (1.0 M imidazole, 1.0 M NaCl, 0.2% Triton-X 100, pH 7.0), 10 L of 10× DTT (50
mM, 5 mM in assay), 40 L of 2-amino-6-mercapto-7-methylpurine riboside (MESG) solution
(1 mM, 400 M in assay), and 5 L of purine nucleotide phosphorylase (PNP) solution (20
U/mL, 1 U/mL in assay). An amount of 5 L of STEP phosphatase (2 M, 100 nM in assay) was
added, and the 96-well assay plate was incubated at 27 °C for 3 min. The coupled assay was
started by addition of 5 L of the appropriate substrate dilution in DMSO (typically 3.00, 1.20,
0.480, 0.192, 0.077, 0.031, 0.012, 0 mM in assay). The plate was then immediately placed into a
spectrophotometric plate reader, and 20 min of kinetic data was obtained (360 nm, 27 °C). The
initial rate data collected were used for Michaelis−Menten kinetic analysis where the Km was
obtained using the substrate−velocity data with the equation v = (Vmax[S])/(Km+[S]).
Determination of inhibitor Ki
Reaction volumes of 100 L were used in 96-well plates. An amount of 65 L of water was
added to each well, followed by 5 L of 20× buffer (1.0 M imidazole, 1.0 M NaCl, 0.2% Triton-
X 100, pH 7.0), 10 L of 10× DTT (50 mM, 5 mM in assay), 5 L of STEP phosphatase (2 M,
100 nM in assay), and 5 L of the appropriate inhibitor dilution in DMSO (with 2- or 3-fold
serial dilutions). The assay plate was incubated for 5 min at 27 °C, at which point the reaction
was started by addition of 10 L of a 10× pNPP substrate (5 mM, 500 M in assay; Km = 745
). The plate was then immediately placed into a spectrophotometric plate reader, and 20 min
of kinetic data was obtained (405 nm, 27 °C). The initial rate data collected were used for
determination of Ki values. For Ki determination, the kinetic values were obtained directly from
nonlinear regression of substrate−velocity curves in the presence of various concentrations of
inhibitor. Assays were run in at least duplicate using the same inhibitor stock solutions.
For the selectivity assays, the Km of pNPP toward each of the enzymes was determined in the
above assay buffer and used for data analysis. For the assays with the dual-specificity MKP5,
76
due to poor turnover of pNPP, the chromogenic substrate 6,8-difluoro-4-methylumbelliferyl
phosphate (DiFMUP) was used instead. In addition, the assay buffer used was a 10× buffer (0.2
M Tris-HCl, 0.2% Triton-X 100, pH 8.0); 5 mM DTT was used as in the other assays.
Expression and purification of STEP
Protein was expressed from BL21(DE3) cells (Invitrogen) which were pre-transformed with
the pGEX-4T-1 vector (GE Healthcare) containing the full length STEP-GST fusion construct.
This pre-transformed stock was provided by the Lombroso group. Transformed bacteria were
grown to an OD600 of 0.8 in LB broth and protein expression was induced by the addition of
400 M isopropyl -D-1-thiogalactopyranoside. After 18 h of expression at 20 °C, cells were
harvested and resuspended in lysis buffer (1× PBS, pH 7.4 with 0.2% Triton X-100). Cell
suspensions were French pressed (×3), and the lysates centrifuged for 40 min at 21,000 × g, and
the cleared lysate loaded onto a glutathione-sepharose column (GE Healthcare). After elution
with reduced glutathione (10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0, 2 mM DTT),
the protein was dialyzed into storage buffer (20 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA) and
concentrated to 13.9 mg/mL (193 M) and was aliquoted appropriately for phosphatase assays.
Cell culture and Western blotting
The Yale University Institutional Animal Care and Use Committee approved all procedures.
Primary cortical neurons were isolated from Sprague−Dawley rat embryos (E18) (Charles River
Laboratories, Wilmington, MA) as previously described.33
Briefly, cells were dissociated with
trypsin, resuspended in Hanks’ balanced salt solution, and then plated on poly-D-lysine-coated
plates (1 × 106 cells/well) in neurobasal medium supplemented with 2% B27 (Invitrogen, San
Diego, CA). Neurons were allowed to grow for 18−21 days at 37 °C in a CO2 incubator.
Compounds to be tested were diluted in DMSO and added to the medium at final concentrations
of 0, 0.1, 1, and 10 M and incubated for 1 h at 37 °C in a CO2 incubator. The percentage of
DMSO remained constant (0.1%) in all wells. After incubation, neurons were lysed in
radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail
(Roche Applied Science, Indianapolis, IN) and phosphatase inhibitors (NaF and Na3VO4).
Protein concentrations of the samples were estimated using the BCA assay kit (Pierce, Thermo
Scientific).
Samples were prepared and resolved by SDS-PAGE, transferred to nitrocellulose membrane,
and incubated with phospho-specific antibodies (anti-pY204/187
ERK1/2, anti-pY402
Pyk2, anti-
pY1472
GluN2B) or total protein antibodies (anti-Pyk2, anti-GluN2B, anti-ERK1/2, anti-GAPDH)
overnight at 4 °C. All antibodies used in this study are listed in Table 3.6. Membranes were
washed and incubated in peroxidase-conjugated secondary antibodies (GE Healthcare,
Waukesha, WI). The immunoreactivity was visualized using a chemiluminescent substrate kit
(Pierce Biotechnology, Rockford, IL) and detected using a G:BOX with the image program
GeneSnap (Syngene, Cambridge, U.K.). All densitometric quantifications were performed using
the Image J (NIH) software.
All data are presented as the mean ± SEM. Differences among multiple groups were
evaluated using one-way ANOVA with Dunnett’s post hoc test using Graph Pad Prism 6
software. For all analyses, a p value of < 0.05 indicated a statistically significant difference.
77
Table 3.6. Primary and secondary antibodies used in this study
Antibody Format Immunogen Host Dilution Source
Anti-pTyr1472
GluN2B
Whole IgG,
unconjugated
Synthetic
phosphopeptide Rabbit 1:1000
Cell Signaling
Technology,
Danvers, MA
Anti-GluN2B Whole IgG,
unconjugated
C-terminus of
mouse GluN2B Rabbit 1:1000
Cell Signaling
Technology
Anti-pTyr402
Pyk2
Whole IgG,
unconjugated
Synthetic
phosphopeptide of
human Pyk2
Rabbit 1:1000 Invitrogen
Anti-Pyk2 IgG2a C-terminus of
human Pyk2 Mouse 1:1000
Cell Signaling
Technology
Anti-pTyr204
ERK1/2
Whole IgG,
unconjugated
Synthetic
phosphopeptide Mouse 1:500
Santa Cruz
Biotechnology
Anti-ERK2 Whole IgG,
unconjugated
C-terminus of rat
p44 MAP Kinase Rabbit 1:20,000
Cell Signaling
Technology
Anti-GAPDH,
clone 6c5
IgG1,
unconjugated
Purified protein
from rabbit muscle Mouse 1:20,000 Millipore
Anti-rabbit
Whole IgG
peroxidase-
conjugated
Rabbit Fc Donkey 1:10,000 Amersham
Biosciences
Anti-mouse
Whole IgG
peroxidase-
conjugated
Mouse Fc Sheep 1:5,000 Amersham
Biosciences
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80
81
Chapter 4. Benzopentathiepins as novel redox-reversible inhibitors of STEP
Abstract: This chapter discusses the discovery and characterization of benzopentathiepins as
redox-reversible inhibitors of STEP. The majority of the chapter focuses on the biochemical
characterization of the benzopentathiepin 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-
amine hydrochloride (TC-2153). It is established that the cyclic polysulfide pharmacophore
forms a reversible covalent bond with the catalytic cysteine in STEP. Several analogs of TC-
2153 are prepared to define not only what is important for inhibition, but also to identify
locations on the molecule that are amenable to diversification for further compound
development. Finally, TC-2153 is shown to be active in cell-based secondary assays and in
animal behavioral models. The majority of this work has been published as a full article (Xu, J.;
Chatterjee, M.; Baguley, T. D.; Brouillette, J.; Kurup, P.; Ghosh, D.; Kanyo, J.; Zhang, Y.; Seyb,
K.; Ononenyi, C.; Foscue, E.; Anderson, G. M.; Gresack, J.; Cuny, G. D.; Glicksman, M. A.;
Greengard, P.; Lam, T. T.; Tautz, L.; Nairn, A. C.; Ellman, J. A.; Lombroso P. J. PLoS Biol.
2014, 8, e1001923).
82
Authorship
This work was conducted in collaboration with Dr. Jian Xu, Dr. Manavi Chatterjee, Dr.
TuKiet Lam and Jean Kanyo. I synthesized all of the compounds and performed all the in vitro
biochemical characterization of the inhibitors including the IC50 determinations, kinact/Ki
determinations, and dialysis and compound stability experiments. Dr. Jian Xu and Dr. Manavi
Chatterjee, researchers in Dr. Paul Lombroso’s research group (Yale School of Medicine),
conducted the cell based assays and animal behavioral studies presented in this chapter. Jean
Kanyo and Dr. TuKiet Lam (Keck Biotechnology Resource Laboratory, Yale) were responsible
for obtaining the LC-MS/MS data.
Introduction
STEP as a therapeutic target
As mentioned in chapter 3, STEP is a phosphatase discovered by our collaborators at the
Yale School of Medicine that has been implicated in many neurodegenerative diseases, such as
Alzheimer’s disease (AD).1 Genetic deletion of STEP has been shown to improve cognitive
function in 3xTg-AD mice in a variety of behavioral models, including the Morris water maze
(Figure 4.1).1a
In the Morris water maze test, the time it takes a mouse to escape a familiar maze
is recorded. If the mouse has normal cognitive faculties, its escape latency will decrease with
subsequent training sessions. AD model mice are impaired in this task (3xTg-AD), but the
genetic deletion of STEP ameliorated this cognitive deficit (double mutant (DM): 3xTg-AD that
are also STEP–/–
). The Lombroso group has been extremely interested in attaining STEP
inhibitors since its discovery because of these positive biological results.
Figure 4.1. Escape latency of AD mouse models showing the 3xTg-AD disease model mice that are also
genetic knockout STEP–/–
(double mutants, DM) have improved cognitive function over the diseased
model mice.1a
Initial high throughput screening results
Parallel to our efforts in chapter 3, the Lombroso research group conducted a HTS to identify
inhibitors of STEP. After screening a library of around 150,000 compounds from the Laboratory
for Drug Discovery in Neurodegeneration (LDDN) library, eight compounds were selected for
83
further characterization based on chemical structure and IC50 values, which ranged between 1
M and 9.7 M, including compound 4.01 (Figure 4.2). Additionally, studies of these
compounds indicated potent inhibition of STEP activity in neuronal cultures and cortical tissue
after intraperitoneal (i.p.) injections in wild-type mice (data not shown).
Figure 4.2. Compound 4.01 was identified as a lead compound from HTS efforts against STEP.
However, following re-synthesis of several of the lead compounds, including compound
4.01, all exhibited essentially no inhibitory activity towards STEP (Figure 4.3). After preparative
HPLC on compound 4.01 and testing the fractions for inhibitory activity, it was discovered that
the there was a highly active contaminant in the commercial samples of the original library
compounds. After isolating this impurity, it was determined that the active component was
elemental sulfur (S8). Encouragingly, elemental sulfur demonstrated good activity in in vitro
enzyme assays, neuronal cultures and cortical tissue after intraperitoneal (i.p.) injections in wild-
type mice (Figure 4.4).
Figure 4.3. Activity of commercial (a) and resynthesized (b) 4.01 against STEP. Upon resynthesis,
compound 4.01 no longer showed appreciable inhibition.
Figure 4.4. Activity of elemental sulfur (S8) against STEP in (a) in vitro enzyme assays (IC50 = 17.2 ± 0.4
nM), (b) neuronal cultures and (c) cortical tissue after i.p. injections in wild-type mice.
84
Benzopentathiepins as attractive target molecules
As a potential drug or tool compound lead, elemental sulfur is a poor compound for two
primary reasons: first, it is very poorly soluble in aqueous solutions; and second, S8 cannot be
modified to improve physicochemical properties, redox activity, binding affinity, or selectivity.
Therefore, we sought to identify more conventional inhibitor structures that would improve
solubility and enable further refinement through analog preparation and evaluation. We
identified the benzopentathiepin core structure 4.03, which is present in a number of natural
products, as the most promising for further investigation (Figure 4.5). Natural products
incorporating the benzopentathiepin core motif have been reported to have antifungal and
antibacterial activity in cell culture as well as cytotoxicity against human cancer cell lines.2
Additionally, amino substituted derivatives such as varacin (4.04) and 8-(trifluoromethyl)-
1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (TC-2153, 4.05) have reasonable solubility
in aqueous solution.3 TC-2153 was designed as an analog to benzopentathiepin containing
natural products by a research team at a Siberian research institute and reportedly has a low level
of acute toxicity (LD50 > 1,000 mg/kg) and was proposed to cross the blood brain barrier as
evidenced by anxiolytic and anticonvulsant effects in mice.4 Finally, from a medicinal chemistry
point-of-view, we imagined that the scaffold present in TC-2153 would allow for modification to
address any physicochemical properties of interest.
Figure 4.5. Polysulfide containing compounds, including TC-2153 (4.05).
Synthesis of TC-2153
The synthesis of TC-2153 began with the literature synthesis of intermediate 4.08 (Scheme
4.1).5 Commercially available 2-chloro-1,3-dinitro-5-(trifluoromethyl)-benzene (4.06) first
undergoes a double SNAr reaction with sodium N,N-dimethyldithiocarbamate to give
benzodithiolone 4.07 in 40% yield. Further attempts to optimize the yield of this reaction were
unsuccessful because upon hydrolysis of the intermediate iminium ion, either dimethyl amine or
the thiophenol can act as a competent leaving group, with only the former leading to the desired
product. Treatment of intermediate 4.07 with excess sodium hydrosulfide (NaSH) yields the
desired benzopentathiepin intermediate 4.08 in 41% yield. In this reaction, the hydrosulfide acts
as both the terminal reductant to reduce the nitro group to the aniline and as a source of sulfur to
form the pentathiepin ring. Although other ring sizes are possible, the 7-membered pentathiepin
is the thermodynamically most stable species and it adopts a chair-like structure.3b
However, in
addition to the desired product, multiple polysulfidic byproducts are also formed in this reaction,
including nitrotrithiol 4.09, and the dimeric structure 4.10, which greatly complicate the isolation
and purification of compound 4.08. By lowering the concentration in the reaction, less of dimer
4.10 is formed, but the nitro compound 4.09 becomes the major product of the reaction (data not
shown). It was first thought that compound 4.09 is an intermediate product, resulting from partial
85
pentathiepin ring formation prior to nitro reduction. However, resubjecting 4.09 to the reaction
conditions yields no desired product. Based upon this result we hypothesized that the reduction
of the nitro group occurs first, followed by pentathiepin ring formation and that nitro reduction
cannot occur in the presence of the trithiol intermediate.
Scheme 4.1. Original synthesis of intermediate 4.08
In order to test this hypothesis, we elected to selectively reduce the nitro group of 4.07 to
provide aniline 4.11, which would then be subjected to the pentathiepin ring formation step
(Scheme 4.2). Adopting a literature procedure,6 we found that reduction of the nitro group
proceeded cleanly upon treatment with tin (II) chloride and concentrated HCl to yield the desired
aniline 4.11 in 90% yield and high purity with only extractive isolation. Subsequent treatment
with NaSH yields the desired intermediate 4.08 in better yield (55% over two steps) than the
original synthesis, and more importantly eliminates the major byproducts that complicated
purification and resulted in impure product. One other parameter that was crucial to minimizing
formation of dimer 4.10 was the proper workup conditions of the reaction. The reaction must be
quenched with concentrated HCl (1 N, 4 N, and even 10 N HCl led to an increase of dimer
formation, data not shown) and the acidic solution must be neutralized by NaHCO3. Quenching
with NaOH, even at 1 N but especially higher concentrations, led to an increase in dimer
formation (data not shown).
Scheme 4.2. Selective reduction of the nitro group in 4.07 to give 4.11 leads to cleaner conversion to 4.08
Further optimization of this selective nitro reduction strategy led to an optimized synthesis of
TC-2153 (Scheme 4.3). The final reduction conditions of ammonium chloride and zinc dust7
were chosen for ease of scalability with typical yields ranging from 85% to 98% for the
reduction step depending on the scale of the reaction. This synthetic sequence eliminates
contaminating byproducts. Additionally, this sequence has been used to generate > 20 gram
quantities of TC-2153 in high purity, which was needed for ongoing animal studies,
pharmacokinetic (PK) studies and analog synthesis (vide infra).
86
Scheme 4.3. Optimized synthetic route for the preparation of multi-gram quantities of TC-2153
Mechanism of STEP inhibition by TC-2153
Enzymatic characterization of inhibition
With TC-2153 prepared, we could begin to answer the question of if and how it may inhibit
STEP. Initial in vitro assays of the compound indicated irreversible inhibition (Figure 4.6). In the
standard inhibition assay, TC-2153 showed potent inhibition with an IC50 of 24.6 ± 0.8 nM
(Figure 4.6a). To evaluate the mode of inhibition, STEP was incubated with TC-2153, the
sample was subjected to dialysis to remove excess inhibitor, and enzyme activity was determined
(Figure 4.6b). After 24 h of dialysis, STEP remained inhibited, establishing that TC-2153 acts as
an irreversible inhibitor under these conditions. Using the progress curve method,8 inhibition was
also found to be irreversible and the second-order rate of inactivation was determined (Figure
4.6c). A kobs was determined for pNPP in the presence of varying initial inhibitor concentrations
(n ≥ 4). Values were then analyzed with non-linear regression to obtain the kinetic constants:
kinact = 0.0176 ± 0.0007 s–1
; Ki = 115 ± 10 nM; kinact/Ki = 153,000 ± 15,000 M–1
s–1
.
Figure 4.6. TC-2153 irreversibly inhibits STEP under standard assay conditions. (a) The IC50 of TC-2153
is 24.6 ± 0.8 nM. (b) Inhibition is not reversed with dialysis of excess inhibitor. (c) The progress curve
method was used to determine the second-order rate of inactivation: kinact/Ki = 153,000 ± 15,000 M–1
s–1
.
Reduced glutathione (GSH) is a ubiquitous reducing agent in cells,9 and may interact with
TC-2153 because of its polysulfide character. Therefore, STEP inhibition was tested with the
addition of thiols (i.e., GSH and DTT) and inhibition was found to be reversible under these
conditions (Figure 4.7). The addition of GSH (1 mM) decreased the inhibitory activity of TC-
2153 by two orders of magnitude in in vitro assays (Figure 4.7a). Inhibition of STEP by TC-2153
is also reversible through the addition of thiol reducing agents as seen by the recovery of STEP
87
activity after pre-inhibition with TC-2153 (Figure 4.7b). In particular, aliquots of STEP were
incubated with DMSO control or TC-2153 and were then added to assay buffer containing 1 mM
GSH, 1 mM DTT, or water control and allowed to incubate for up to 1 h prior to testing for
enzymatic activity. STEP activity was rapidly recovered by both reductants, with DTT showing a
greater recovery of activity (75% recovery after 1 h, where DMSO control represents 100%
activity) compared to GSH (29% recovery after 1 h).
Figure 4.7. Inhibition of STEP by TC-2153 is reversible through the addition of thiol reducing agents. (a)
The IC50 of TC-2153 is 8.8 ± 0.4 M with addition of 1 mM GSH to the assay. (b) STEP activity is
recovered from pre-inhibited STEP by treatment with thiol reducing agents.
These results suggested an oxidative mechanism for the inhibition of STEP, similar to the
redox-reversible regulation of PTPs discussed in chapter 1. An early hypothesis was that TC-
2153 was decomposing or otherwise generating reactive oxygen species (ROS) under the assay
conditions, but these mechanisms for STEP inhibition were ruled out through a series of
experiments. First, by monitoring the 19
F NMR signal of TC-2153 (details in experimental
section) it was established that TC-2153 was stable and did not degenerate in the assay
conditions. Moreover, upon addition of catalase or superoxide dismutase to the in vitro assay
(Table 4.1), there was no change in inhibition, indicating that TC-2153 is not acting through
generation of ROS.
Table 4.1. In vitro inhibition of STEP by TC-2153
Conditions IC50 (nM)
no additivea 24.6 ± 0.8
+ catalaseb,c
26.2 ± 0.6
+ SODb,d
18.6 ± 0.8
+ catalase + SODb,c,d
24.5 ± 4.2
+ GSHb,e
8,790 ± 430 aMean ± S.D. (n = 4).
bMean ± S.D. (n = 2).
c80 U/mL catalase.
d100 U/mL
superoxide dismutase (SOD). e1 mM reduced glutathione (GSH).
LC-MS/MS characterization of inhibition
In order to further elucidate the inhibition of STEP by TC-2153, LCMS analysis was
performed to determine the intact protein mass of STEP and STEP+TC-2153. The intact protein
analyses suggest a covalent adduct to STEP. Although we were able to obtain the accurate mass
for STEP, we were unable to resolve the heterogeneous mixture of intact STEP+TC-2153 and its
covalent adducts with sufficient accuracy to fully interpret the results.
88
Therefore, we next used high-resolution tandem mass spectrometry to focus upon whether
TC-2153 might modify the active site cysteine of STEP. For these experiments, we used WT
STEP as well as a STEP mutant in which the catalytic cysteine was changed to serine. Greater
than 90% of the primary amino acid sequences were identified by LC-MS/MS for WT STEP or
for the STEP mutant, following in-gel tryptic digestion of STEP from non-denaturing (native)
preparations. We initially analyzed the catalytic cysteine at position 472 of STEP in the absence
of TC-2153 and found a disulfide bridge between Cys465
and Cys472
that presumably forms
following tryptic digestion given that Cys465
and Cys472
are > 20 Å apart from each other in the
3-dimensional X-ray crystal structure of STEP.10
This modification was not observed when the
catalytic site cysteine (Cys472
) was mutated to serine. Incubation of WT STEP with TC-2153
resulted in the presence of a de novo trisulfide within the Cys465
/Cys472
bridge. Consistent with
covalent modification by TC-2153, this modification that was not observed for WT STEP alone
or when the catalytic site cysteine (Cys472
) was mutated to serine (Figure 4.8). The precursor
monoisotopic mass of the trisulfide-containing peptide had a mass error of 4 ppm (~0.011 Da)
based on theoretical mass calculation, which is within the 5 ppm external mass calibration
expected for MS/MS data collected by the linear ion trap instrument used.
Figure 4.8. Detection of trisulfide bridge formation between C
465 and C
472. The peptide sequence in (a)
illustrates the trisulfide bridge along with the b and y-ions assignments detected in the MS/MS
fragmentations spectrum. Part (b) compares the 3D elution profile of the trisulfide peptide (mass =
2746.242 Da). The trisulfide bridge (modified) peptide is only detected in the WT STEP in the presence
of TC-2153. The corresponding disulfide (non-modified) peptide (mass = 2714.254Da) was detected in
WT STEP.
TC-2153 was the only exogenous source of S atoms in these samples. Although the direct
modification, whether whole or partial molecule attachment, cannot be inferred from this data,
these results indicate that the active site cysteine is likely modified by TC-2153 and suggest that
following tryptic digestion a sulfur atom from the benzopentathiepin core is retained giving rise
to the trisulfide which was identified by mass spectrometry.
Preparation of TC-2153 analogs for STEP inhibition
We were next interested in synthesizing derivatives of TC-2153 to be evaluated for inhibition
of STEP. Given the promising biological properties of TC-2153 (vide infra), we were motivated
to define the essential characteristics of the molecule necessary for enzyme inhibition (Figure
4.9). First, we wished to explore the electronic effects of the inhibitor core to assess the
importance of electronics on the redox-reversible inhibition of STEP. Secondly, we wanted to
89
discover sites on the molecule tolerant of substitution to enable modulation of physicochemical
properties such as solubility, for the introduction of reporter groups, or for the introduction of
functionality to facilitate pull down assays and proteomic analysis.
Figure 4.9. Proposed SAR study of TC-2153.
TC-2153 analog synthesis
To prepare the analogs, we first investigated their synthesis using the sequence reported by
Kulikov et. al. (Scheme 4.4),5 which was introduced in Scheme 4.1. Elevated temperatures were
required for the SNAr reactions with sodium dimethyldithiocarbamate when the less activated
substrates 4.12 and 4.13 containing a simple methyl or proton substituent at the R position were
used, with lower temperatures resulting only in a single nucleophilic substitution of the chloride.
Consistent with a previous report,11
treatment of the intermediate nitrodithiolones 4.14 and 4.15
with NaSH yielded little to no desired product and only nitrotrithiol byproducts were formed in
any appreciable yield (e.g., compound 4.09). As was discussed above for the synthesis of TC-
2153, by first reducing the nitro group to the free anilines, the desired synthetic intermediates
could be obtained (Scheme 4.5), and the synthesis of TC-2153 analogs 4.20 and 4.21 could be
completed.
Scheme 4.4. Attempted synthesis of benzopentathiepin compounds 4.16 and 4.17
Scheme 4.5. Completed synthesis of TC-2153 analogs 4.20 and 4.21
Analog 4.24, which does not contain an amine, was synthesized using an analogous
procedure (Scheme 4.6). 2-Chloro-1-nitro-4-(trifluoromethyl)benzene 4.22 was treated with
sodium dimethyldithiocarbamate at elevated temperature to yield the dithialone 4.23. Treatment
with NaSH in DMSO yielded the analog 4.24. The amine provides a convenient handle for
potentially adding solubilizing functionality or chemical labels, which has the potential of being
90
introduced either by alkylation to maintain the basicity of the amine or by acylation. Acylation of
intermediate 4.08 according to literature precedent with either acetyl chloride or trifluoroacetic
anhydride yielded analogs 4.25 and 4.26 respectively (Scheme 4.7).4 Reductive amination of
4.08 with acetaldehyde or benzaldehyde provided N-ethyl and N-benzyl analogs 4.27 and 4.28
(Scheme 4.8).
Scheme 4.6. Synthesis of TC-2153 analog 4.24
Scheme 4.7. Synthesis of TC-2153 analogs 4.25 and 4.26 through acetylation of 4.08
Scheme 4.8. Synthesis of TC-2153 analogs 4.27 and 4.28 through reductive amination of 4.08
Inhibition of STEP by TC-2153 analogs
The inhibitory activity of the analogs was determined against STEP both in the presence and
the absence of 1 mM GSH (Table 4.2). Because of the differential results with TC-2153 (Figure
4.7a), we thought it was prudent to investigate the inhibitor activity under both of these
conditions, as GSH may interact with the inhibitors differentially. In the absence of GSH the
most potent inhibitor in the series was the simple 7-(trifluoromethyl)-benzopentathiepin 4.24 (10
± 1 nM). However, with the absence of the amine substituent, the solubility and thus future
utility of 4.24 is limited. With a benchmark IC50 of 24.6 ± 0.8 nM, TC-2153 (4.05) remains one
of the most potent analogs in this series, along with the other aniline hydrochloride salts 4.20 and
4.20 (25 ± 7 nM) and 4.21 (32 ± 3 nM). Trifluoroacetamide derivative 4.25 also showed good
potency (24 ± 1 nM), while the less electron deficient acetamide derivative 4.26 showed a two-
fold decrease in inhibitory activity (49 ± 2 nM). Alkylated anilines 4.27 and 4.28, which are
slightly less electron deficient than the parent aniline, showed a marked decrease in potency.
91
Interestingly, the byproducts generated in the synthesis of TC-2153 (4.09 and 4.29) also showed
activity against STEP at decreased potency.
When GSH was added to the assay mixture, the same trends generally held, with the acylated
derivatives performing comparably to the free anilines and the alkylated derivatives having
slightly decreased potency. In all cases, there was a decrease in potency by 2–3 orders of
magnitude when GSH was present in the assay.
Table 4.2. Inhibition of STEP in vitro in the presence and absence of GSH
entry compound R1 R
2 IC50 (nM)
a,b IC50 (M)
a,c
1 4.05 (TC-2153) CF3 NH3Cl 24.6 ± 0.8d
8.8 ± 0.4d
2 4.24 CF3 H 10 ± 1 17 ± 2
3 4.20 CH3 NH3Cl 25 ± 7 17 ± 1
4 4.21 H NH3Cl 32 ± 3 33 ± 4
5 4.25 CF3 NHCOCF3 24 ± 1 15 ± 2
6 4.26 CF3 NHAc 49 ± 2 24 ± 1
7 4.27 CF3 NHEt 59 ± 9 > 50
8 4.28 CF3 NHBn 78 ± 3 27 ± 5
9 4.29 -- -- 33 ± 1 20 ± 1
10 4.09 -- -- 145 ± 6 34 ± 4 aAssays were performed in duplicate (mean ± S.D.).
bAssays contained no GSH.
cAssays contained 1 mM
GSH. dAssays were performed in quadruplicate (mean ± S.D.)
Because the inhibition of STEP by TC-2153 was shown to be irreversible in the absence of
GSH (Figure 4.6), we next determined the second order rate of inactivation (kinact/Ki) for all the
inhibitors under these conditions using the progress-curve method (Table 4.3).8 These results
demonstrate that the methyl (4.20) and unsubstituted (4.21) derivatives are about three times less
potent than trifluoromethyl substituted TC-2153 (4.05). Additionally, the alkylation in
derivatives 4.27 and 4.28 was further confirmed to not be beneficial. Even though their Ki values
indicated that binding is not as favored, acylation of the scaffold (4.25 and 4.26) resulted in
inhibitors with higher potency than TC-2153 because of their much higher kinact values relative to
the non-acylated parent structure 4.05.
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Table 4.3. Second-order rates of inactivation of TC-2153 analogsa
entry compound kinact/Ki (M–1
s–1
) Ki (nM) kinact (s–1
)
1 4.05 (TC-2153) 153,000 ± 15,000 115 ± 10 0.0176 ± 0.0007
2 4.24 135,000 ± 18,000 109 ± 14 0.0147 ± 0.0007
3 4.20 41,000 ± 5,300 116 ± 14 0.0048 ± 0.0002
4 4.21 52,200 ± 8,000 183 ± 25 0.0095 ± 0.0006
5 4.25 161,000 ± 26,000 235 ± 33 0.0378 ± 0.0027
6 4.26 193,000 ± 49,000 123 ± 29 0.0236 ± 0.0022
7 4.27 53,500 ± 8,800 230 ± 34 0.0123 ± 0.0009
8 4.28 33,200 ± 2,400 175 ± 11 0.0058 ± 0.0002
9 4.29 138,000 ± 23,000 124 ± 19 0.0171 ± 0.0011
10 4.09 98,000 ± 20,000 210 ± 38 0.0205 ± 0.0018 aAssays were performed in quadruplicate (mean ± S.D.) and in the absence of GSH.
TC-2153 activity in cell-based secondary assays and in vivo
TC-2153 activity in cortical neurons and in vivo
The activity of TC-2153 was next tested in secondary cell-based assays. Cortical neurons
were treated for 1 h with TC-2153 and Tyr phosphorylation levels of residues that STEP
dephosphorylates on GluN2B (Y1472
), Pyk2 (Y402
), and ERK1/2 (Y204/187
) were determined.
There was a significant increase in the Tyr phosphorylation of all three STEP substrates (Figure
4.10a) (1 M dose: pGluN2B: 2.07 ± 0.15, p < 0.001; pPyk2: 1.81 ± 0.21, p < 0.001; pERK1/2:
2.39 ± 0.18, p < 0.001). The decrease in Tyr phosphorylation in the presence of the highest dose
of TC-2153 (10 M) may be due to off-target effects on positive regulatory PTPs. Similar
inverted-U dose-response curves on Tyr phosphorylation of direct PTP targets has been observed
in previous work with PTP inhibitors.12
It was then determined whether TC-2153 inhibited STEP activity in WT mice in vivo. Six-
month old male mice (C57BL/6) were injected with vehicle or TC-2153 (1, 3, 6, 10 mg/kg, i.p.)
and cortices were removed and processed 3 h post injection. TC-2153 led to a significant
increase in the Tyr phosphorylation of GluN2B, Pyk2, and ERK1/2 (at 10 mg/kg: pGluN2B:
1.66 ± 0.28, p < 0.01; pPyk2: 1.80 ± 0.30, p < 0.05; pERK1/2: 2.52 ± 0.16, p < 0.01) (Figure
4.10b). Together, these results demonstrate that TC-2153 increases the Tyr phosphorylation of
three STEP substrates in intact neurons in culture and in vivo in the cortex of WT mice.
TC-2153 specificity in vivo
To address possible off-target inhibition by TC-2153 in cells, cortical cultures from either
WT or STEP KO mice were treated with TC-2153. Similar to the rat neuronal cultures, there was
an observed increase in the Tyr phosphorylation of STEP substrates in WT mouse cortical
neurons (Figure 4.11, black bars). Consistent with previous findings,13
STEP substrates have
higher basal Tyr phosphorylation levels in STEP KO cultures. TC-2153 failed to increase the
phosphorylation of STEP substrates in the KO cultures (Figure 4.11, grey bar with 0.1 M and 1
M). To exclude a possible ceiling effect, the generic tyrosine phosphatase inhibitor, sodium
orthovanadate (Na3VO4), was added and the Tyr phosphorylation of these substrates was further
93
increased. These results suggest that TC-2153 is relatively specific towards STEP compared to
the generic tyrosine phosphatase inhibitor sodium orthovanadate.
Figure 4.10. (a) Cortical neuronal cultures were treated with TC-2153 and vehicle (0.05, 0.1, 1 and 10
M) for 1 h. Phosphorylation of GluN2B (Y1472
), Pyk2 (Y402
) and ERK1/2 (Y204/187
) were significantly
higher after treatment of cultures with TC-2153 (*p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA
with post hoc Bonferroni test). Data represent the phospho-signal normalized to total protein and then to
GAPDH (mean + s.e.m., n = 4). (b) C57BL/6 mice (3–6 months) were injected with TC-2153 (i.p., 1, 3,
6, 10 mg/kg) and were sacrificed 3 h later. Cortices were micro-dissected and lysates spun down to P2
fraction and prepared for western blotting. Tyrosine phosphorylation status was probed with phospho-
specific antibodies to pGluN2B: Tyr1472
, pPyk2: Tyr402
and pERK1/2: Tyr204/187
(*p < 0.05; **p < 0.01;
one-way ANOVA with post hoc Bonferroni test). Data represent the phospho-signal normalized to the
total substrate protein signal and then to GAPDH as a protein expression control (mean + s.e.m., n = 3).
Figure 4.11. TC-2153 failed to increase tyrosine phosphorylation of STEP substrates in STEP KO
cortical neurons. WT and STEP KO cultures were treated with TC-2153 (0.1 and 1 M), vehicle (0.1%
DMSO) or sodium orthovanadate (Na3VO4, 1 mM) for 1 h. Phosphorylation of GluN2B Y1472
, Pyk2 Y402
and ERK1/2 Y204/187
was normalized to total substrate protein level and then to GAPDH as a protein
expression control (*p < 0.05, **p < 0.01 one-way ANOVA with post hoc Bonferroni test, compared with
veh-treated controls, n = 4).
Additionally, there are three highly related PTPs (STEP, HePTP, and PTP-SL) that all
dephosphorylate ERK1/2.14
Only STEP is found in cortex, while HePTP is present in spleen, and
PTP-SL is present in cerebellum, both tissues that lack STEP. In addition, ERK1/2 and Pyk2 are
dephosphorylated by other tyrosine phosphatases outside of the CNS. To further probe this
94
apparent specificity, WT and STEP KO mice were injected with TC-2153 or vehicle, and the Tyr
phosphorylation of ERK1/2 (Y204/187
) and Pyk2 (Y402
) in different tissues was determined (Figure
4.12). There was a significant increase observed in pERK1/2 and pPyk2 in the frontal cortex and
hippocampus, tissues that contain STEP, but not in the cerebellum or in all tissues tested outside
the brain. These results suggest that TC-2153 does not target homologous PTPs known to
dephosphorylate ERK1/2 and Pyk2 when tested in vivo.
Figure 4.12. TC-2153 increased the phosphorylation of ERK1/2 Y
204/187 and Pyk2 Y
402 in frontal cortex
(a) and hippocampus (b), tissues that contain STEP, but not in cerebellum, spleen, kidney or pancreas (c–
f), all tissues that do not have STEP. Mice were injected i.p. with TC-2153 (10 mg/kg; n = 4) or vehicle (n
= 4) and were sacrificed 3 h later. Changes are expressed as the mean ± s.e.m. of pERK1/2 and pPyk2
normalized to total substrate protein level and then to GAPDH as a protein expression control (*p < 0.05,
**p < 0.01; two-way ANOVA followed by Tukey’s H.S.D. test).
TC-2153 reduces cognitive deficits in 3xTg-AD mice
Next, the efficacy of TC-2153 to reverse cognitive deficits in an AD mouse model was tested
in the reference memory version of the Morris water maze (MWM). A three-way ANOVA
analysis revealed a significant genotype × treatment × training day interaction (p < 0.05). Daily
injection of TC-2153 3 h prior to training reversed memory deficits in 3xTg-AD mice on days 5
and 6 of the acquisition phase (p < 0.01) (Figure 4.13a). The longer escape latency of 3xTg-AD
mice injected with vehicle was not attributed to slower swimming speed since no significant
95
differences were found between groups (p > 0.05; two-way ANOVA) (Figure 4.13b). To confirm
memory status, the number of entries in a circular zone located around the previous platform
location (target zone) and in the opposite quadrants was evaluated during the probe trial 24 h
after the last acquisition day. A three-way ANOVA analysis revealed a significant genotype ×
treatment × quadrant interaction (p < 0.004). The 3xTg-AD mice treated with TC-2153 spent as
much time as WT mice in the target zone, while AD mice injected with vehicle showed no
preference for the target zone (Figure 4.13c). All groups had similar escape latencies during the
cued trial when the platform was visible, indicating the absence of sensorimotor or motivational
deficits to escape from water (WTVeh: 15.1 ± 1.7 s; WT-TC: 15.6 ± 1.7 s; AD-Veh: 15.3 ± 3.0 s;
AD-TC: 16.0 ± 2.3 s; mean ± s.e.m.; p < 0.05; two-way ANOVA).
Figure 4.13. WT and 3xTg-AD mice (male, 6-months old) were treated with vehicle or TC-2153 (10
mg/kg, i.p.) and tested MWM tasks. (a) The 3xTg-AD mice injected with vehicle (n = 6) showed longer
escape latency before finding the hidden-platform (3 trials/day; 60s; 30m intertribal interval) when
compared to AD mice treated with TC-2153 (n = 7) or WT mice injected with vehicle (n = 12) or TC-
2153 (n = 13) (three-way ANOVA). *,+ represents a statistical significant variation between AD-Veh
mice and AD-TC or WT-Veh, respectively. (b) Swim speed at each training day was not significantly
different between groups (three-way ANOVA). (c) Number of entries in a circular zone positioned around
the previous platform location and in the opposite quadrants. *represents a statistical significant variation
between AD-TC mice and other groups for the target quadrant. +indicates a difference for the target and
opposite quadrant within each group. Data are mean ± s.e.m. *,+p < 0.05, **,++p < 0.01, ***,+++p <
0.01.
Additionally, TC-2153 treated 6-month old AD 3xTg-AD mice showed significant increases
in performance in the Y-maze (used to evaluate spatial working memory function), open field
and novel object recognition tests (data not shown). Taken together, these results demonstrate
that TC-2153 significantly improved cognitive functioning in mouse models for AD.
Conclusions
After identifying the benzopentathiepin TC-2153 (4.05) as an attractive molecule for
potential STEP inhibition, a reliable and scalable synthesis was developed to access TC-2153 in
high purity and in quantities needed for animal studies. Once synthesized, inhibition of STEP by
TC-2153 was characterized as a redox-reversible mode of inhibition where a sulfur atom from
the pentathiepin ring modified the catalytic Cys. It was also determined that this oxidative
inhibition was not due to generation of ROS.
Additionally, a series of analogs of TC-2153 has been prepared and characterized in in vitro
enzyme assays. Importantly, these results demonstrate that the trifluoromethyl group contributes
96
to the potency of the molecule and that other modifications are tolerated. Most importantly,
acylation of the aniline is accommodated and could provide a site for introducing reporter groups
or functionality for pull down and proteomic applications.
Moreover, TC-2153 was demonstrated to be active in secondary neuronal cortical culture
assays and in vivo. The inhibition in vivo is also selective for tissues that contain STEP and
highly homologous PTPs are not affected in vivo. Importantly, TC-2153 is efficacious in animal
behavioral models, as exemplified by the Morris water maze test where 3xTg-AD mice that were
treated with TC-2153 demonstrated a significant increase in cognitive function. TC-2153 is
currently undergoing PK studies at 7th
Wave Laboratories (Chesterfield, MO). In addition,
animal behavioral studies have progressed to aged Parkinsonian rhesus monkeys (Jay Schneider,
Thomas Jefferson University) and initial results indicate that TC-2153 is able to improve
cognitive impairment in these studies as well.
Experimental
General materials and methods
Synthetic methods
Unless otherwise noted, all reagents and solvents were obtained from commercial suppliers
and used without further purification. Diethyl ether (Et2O) and CH2Cl2 were passed through a
column of activated alumina (type A2, 12 × 32, Purify Co.) under nitrogen pressure immediately
prior to use. All 1H,
19F and
13C NMR spectra were obtained at ambient temperature on a Bruker
AVB-400 or AVB-500 spectrometer. NMR chemical shifts are reported in ppm relative to CHCl3
(7.26), or DMSO (2.50) for 1H, trifluoroacetic acid (−76.55) for
19F, and CDCl3 (77.16), DMSO-
d6 (39.52) or CD3OD (49.00) for 13
C. Mass spectrometry (HRMS, ESI, GCMS) are reported in
m/z. Chromatography was performed with SiliCycle SiliaFlash® P60 230–400 mesh silica gel.
Reversed-phase purifications were conducted with a Teledyne Isco CombiFlash Rf system
equipped with HP C18 gold cartridges. Unless otherwise noted, product yields are not optimized.
Melting points were recorded on an Electrothermal melting Point Apparatus and are uncorrected.
Enzymatic assays were carried out on a BioTek Synergy 2 Multi-Mode Microplate Reader.
Bioassays
p-Nitrophenyl phosphate (pNPP), 2-(N-morpholino)ethanesulfonic acid (MES), sodium
orthovanadate (Na3VO4), ATP, and all buffer components were purchased from Sigma-Aldrich
(St. Louis, MO). Malachite Green reagent kit was purchased from Bioassay system (Hayward,
CA). 6,8-Difluoro-4-methylumbelliferyl phosphate (DiFMUP) and EnzChek phosphatase assay
kit were purchased from Invitrogen (Carlsbad, CA). For some of the biochemical experiments,
WT TAT-STEP46 and TAT-STEP46 (C to S) proteins were produced and purified using standard
procedures.15
97
Ethics statement
The Yale University Institutional Animal Care and Use Committee approved all proposed
use of animals. All animal work was carried out in strict accordance with National Institutes of
Health (NIH) Guidelines for the Care and Use of Laboratory Animals.
Synthesis of TC-2153
Compound 4.07. Modifying a known literature procedure,
5 a 1 L round bottomed flask was
charged with a magnetic stir bar, 135 g (0.500 mol) of 2-chloro-1,3-dinitro-5-(trifluoromethyl)-
benzene 4.06 and 70 mL of DMSO. The reaction flask was suspended in an ambient temperature
water bath. A solution of sodium dimethyldithiocarbamate dihydrate (89.7 g, 0.500 mol) in
DMSO (200 mL) was added dropwise via cannula over 2 h. After addition, the reaction mixtures
was stirred for 1 h in the water bath and was quenched by addition of 400 mL of water. The
reaction mixture was transferred to a larger flask and was diluted to 2 L with water, then was
extracted with CH2Cl2 (3 × 1 L and then 3 × 500 mL). The combined organic layer was
condensed to approximately 2 L, washed with brine (1 × 1 L), and dried by stirring over MgSO4
for 20 min. The remaining solvent was removed on a rotary evaporator. The resulting solid was
purified by SiO2 flash chromatography using 4 L of SiO2 and was dry-loaded onto the column
with approximately 200 mL of SiO2. The product was eluted first with 9:1 and then 4:1
hexanes:EtOAc to yield the product as a light orange solid (61.4 g, 43.6%). Analytical data
correlates with the published report.5
Original synthesis of compound 4.08 with byproducts 4.09 and 4.10. Following a
literature report,5 a 100 mL round bottomed flask was charged with a magnetic stir bar and 4.07
(1.75 g, 6.20 mmol). DMSO (25 mL, 0.25 M) was added to dissolve the starting material and
2.30 g (31.0 mmol, 5 equiv) of NaSH-H2O was added to the reaction as a solid in one portion.
The reaction mixture was stirred for 18 h at ambient temperature. To quench the reaction, 20 mL
of concentrated HCl was added, and the mixture was stirred at ambient temperature for 20 min.
The reaction mixture was neutralized with 10 mL of 10 N NaOH, and solid NaHCO3 to pH 8.0,
and was extracted with EtOAc (2 × 50 mL). The organic layer was washed with NaHCO3 (70
mL) and dried over MgSO4. Solids were removed by filtration and the volatile components were
removed via rotary evaporation. The crude residue was purified via SiO2 flash column
chromatography with an eluent of 6:1 hexanes:MTBE. Compound 4.08 was isolated as an orange
oil (808 mg, 41%). The analytical data correlates with the published report.5 Also isolated from
this reaction were side products 4.09 (658 mg, 37%) and 4.10 (78 mg, 5%). 4.09: red solid, m.p.
113–116 °C (lit.16
110–111 °C). 1H NMR (500 MHz, CDCl3): δ 8.24 (d, J = 1.7 Hz, 1H), 7.82 (d,
J = 1.7 Hz, 1H); 19
F NMR (376 MHz, CDCl3): δ –62.91; 13
C NMR (126 MHz, CDCl3): δ 146.67,
98
146.37, 146.08, 130.80 (q, JCF = 34.8 Hz), 124.18 (q, JCF = 3.4 Hz), 122.62 (q, JCF = 273.1 Hz),
121.32 (q, JCF = 4.0 Hz). GCMS (m/z): 285. 4.10: yellow solid, m.p. 146–152 °C (dec). 1H NMR
(400 MHz, CDCl3): δ 7.31 (d, J = 1.8 Hz, 2H), 6.56 (d, J = 1.8 Hz, 2H), 4.77 (s, 4H). HRMS-ESI
(m/z): [M+H]+ calcd. for C14H9F6N2S6, 510.8989; found, 510.8982.
Compound 4.11. The procedure is based on an existing literature procedure with some
modifications.7 A 2 L round bottomed three-neck Morten flask was charged with 300 mL of
water, 300 mL of EtOH, 214 g of zinc metal (3.27 mol, 15 equiv), 93.3 g (1.74 mol, 8 equiv) of
ammonium chloride and was equipped with a mechanical stirrer. The reaction flask was placed
into an ice water bath. Nitro compound 4.07 (61.3 g, 0.218 mol) was added as a solid portion-
wise with mechanical stirring over 15 min. The ice bath was removed, and the reaction solution
was allowed to warm to ambient temperature with mechanical stirring for 24 h. The reaction
mixture was neutralized with the addition of 100 mL of saturated aqueous NaHCO3 and was
filtered through a fritted funnel to remove solids. The filter cake was washed with water (500
mL) and CH2Cl2 (1 L). The layers were separated and the water layer was extracted with CH2Cl2
(3 × 500 mL). The combined organic layers were condensed to 1 L, washed with brine (1 L), and
dried by stirring over MgSO4 for 20 min. The solvent was removed on a rotary evaporator,
yielding 45.8 g (83.5%) of 4.11 as an off-white solid, m.p. 148–151 °C. 1H NMR (500 MHz,
CDCl3): d 7.19 (d, J = 1.5 Hz, 1H), 6.88 (d, J = 1.5 Hz, 1H), 3.98 (s, 2H); 19
F NMR (376 MHz,
CDCl3): d –62.85; 13
C NMR (126 MHz, CDCl3): d 188.20, 141.63, 134.16, 130.61 (q, JCF = 33.0
Hz), 123.90 (q, JCF = 272.7 Hz), 121.05, 110.28 (q, JCF = 4.1 Hz), 109.84 (q, JCF = 3.7 Hz).
HRMS-ESI (m/z): [M+H]+ calcd for C8H5F3NOS2, 251.9759; found, 251.9163.
Compound 4.08. This reaction was performed on 15 g batches to maintain a reasonable
solvent volume during the extraction steps (vide infra). A 2 L round bottomed three-neck Morten
flask was charged with 22.3 g of NaSH-H2O (300 mmol, 0.5 M final concentration), 500 mL of
DMSO, was equipped with a mechanical stirrer and was placed in an ambient temperature water
bath. Aniline 4.11 (15.1 g, 60.1 mmol) was dissolved in 100 mL of DMSO and was added to the
NaSH suspension dropwise via cannula with positive N2 pressure over 1 h with mechanical
stirring. The reaction mixture was stirred at ambient temperature under N2 for 16 h and was then
placed in an ice water bath. The reaction was quenched with 50 mL of concentrated HCl and the
resulting mixture was stirred for 45 min in the cold water bath. The reaction was neutralized to
pH 8.0 with NaHCO3, first with 50 mL of a saturated aqueous solution, then with solid NaHCO3.
The resulting mixture was diluted to 4 L with water, then was separated into two fractions of 2 L
each. Each fraction was extracted with CH2Cl2 (4 × 1 L). The combined organic layer is
concentrated to 2 L and was washed with saturated NaHCO3 (1 L), then with brine (1 L), then
was dried over Na2SO4 with stirring for 20 min. The solids were removed by filtration, and the
volatile components were removed with a rotary evaporator with an ice water bath. The crude
99
residue was purified via SiO2 flash chromatography with an eluent of 6:1 then 4:1
hexanes:MTBE to yield 4.08 in two fractions. Other solvent systems (hexanes:CH2Cl2,
hexanes:EtOAC, pentane:ether) were unsuccessful in separating the product from the very close
minor dimer impurity 4.10. Pure product was isolated as a yellow oil (8.49 g, 44%), and a second
fraction of product (7.83 g) was isolated as an orange oil, which contains a small amount of
byproduct 4.10 (<10%) and can be purified further. The NMR data of the purified product
correlates with the published data.5
TC-2153 (4.05). Free aniline 4.08 (23.3 g, 72.8 mmol) was dissolved in Et2O (220 mL, 0.33
M) in a 1 L glass beaker with a stir bar. Concentrated HCl (9.5 mL, 110 mmol, 1.5 equiv) was
added dropwise and a yellow precipitate formed. The mixture was stirred at ambient temperature
for 90 min, at which point the solid product 4.05 was isolated via filtration. Drying over vacuum
yielded 18.28 g (70%) of the pure product as a light yellow solid. The filtrate was collected,
concentrated, dissolved in 50 mL of Et2O, acidified with 1 mL of concentrated HCl and filtered
to acquire a second crop of solid. The solid was washed with cold ether to remove any orange
color, providing an additional crop of pure product (2.09 g, 8%), m.p. 142–147 °C (dec). 1H
NMR (400 MHz, DMSO-d6): δ 9.40–7.20 (br s, 3H), 7.17 (d, J = 2.0 Hz, 1H), 7.13 (d, J = 2.0
Hz, 1H); 19
F NMR (376 MHz, DMSO-d6): δ –62.43; 13
C NMR (126 MHz, DMSO-d6): δ 153.40,
146.21, 131.16 (q, JCF = 32.1 Hz), 124.75, 123.02 (q, JCF = 273.3 Hz), 117.29, 113.82 (q, JCF =
4.8 Hz). The analytical data of the purified product correlates with the published data.5
Synthesis of TC-2153 analogs
Analogs 4.25 and 2.26 were prepared from 4.08 following literature methods.4
Compound 4.14. Modifying a literature procedure,
5 commercially available 2-chloro-5-
methyl-1,3-dinitrobenzene 4.12 (1.08 g, 5.00 mmol) and sodium dimethyldithiocarbamate
dihydrate (896 mg, 5.00 mmol) were dissolved in 10 mL of DMSO and sealed in a microwave
vial with a stir bar. The reaction mixture was heated in a microwave reactor at 150 °C for 1 h.
After cooling to ambient temperature, the mixture was diluted to 100 mL with water followed by
extraction with CH2Cl2 (3 × 100 mL). The combined organic layer was washed with water (3 ×
150 mL), and brine (150 mL), and dried over MgSO4. Solids were removed by filtration and the
volatile components were removed via rotary evaporation to yield the crude product 4.14 as a
dark orange solid (644 mg, 57%), which was taken on to the next step without purification.
100
Compound 4.18. Reduction was achieved following the procedure for 4.11 beginning with
190 mg of the crude product 4.14 and 359 mg of NH4Cl and 817 mg of zinc metal. The
procedure yielded 110 mg (67%) of 4.18 as a yellow solid, m.p. 140–141 °C. 1H NMR (400
MHz, CDCl3): δ 6.78–6.76 (m, 1H), 6.50–6.48 (m, 1H), 3.71 (br s, 2H), 2.29 (s, 3H); 13
C NMR
(151 MHz, CDCl3): δ 189.91, 140.91, 138.02, 132.99, 114.50, 114.26, 114.08, 21.45. HRMS-
ESI (m/z): [M+H]+ calcd. for C8H8NOS2, 198.0042; found, 198.0034.
Analog 4.20. Compound 4.20 was generated following the procedures TC-2153 above.
Treatment of 19 mg (0.10 mmol) of 4.18 with 37 mg (0.50 mmol) of NaSH-H2O in DMSO (1
mL) yielded 12 mg of the intermediate free amine 4.16 as an orange residue after purification on
SiO2 column chromatography. This material was immediately converted to the desired HCl salt
via treatment with 1.5 equiv of HCl in Et2O to yield 4.20 (10 mg, 33% over two steps) as an off-white solid, m.p. 164–168 °C (dec).
1H NMR (400 MHz, DMSO-d6): δ 7.16–6.69 (br s, 3H),
6.81 (d, J = 1.9 Hz, 1H), 6.62 (d, J = 1.9 Hz, 1H), 2.16 (s, 3H); 13
C NMR (151 MHz, CD3OD): δ
147.81, 144.76, 140.61, 135.12, 132.57, 125.52, 21.05. HRMS-ESI (m/z): [M–Cl]+ calcd. for
C7H8NS5, 265.9255; found, 265.9248.
Compound 4.15. The procedure for the formation of 4.15 was followed with 1.102 g (5.00
mmol) of 2-chloro-1,3-dinitrobenzene 4.13. Purification by filtration of the intermediate through
a SiO2 plug (3:1 pentane/Et2O) yielded the product as a dark yellow solid (509 mg, 48%), which
was taken onto the next step without further purification.
Compound 4.19. Reduction was achieved through a modified literature procedure.
6 A 100
mL round bottomed flask was charged with 500 mg (2.00 mmol) of the crude product 4.15, 1.14
g (6.00 mmol, 3.0 equiv) of SnCl2 and a magnetic stir bar. Ethanol (15 mL) and concentrated
HCl (15 mL) were added to the reaction flask, which was placed in a preheated 40 °C oil bath.
The reaction mixture was refluxed under N2 for 18 h. The reaction flask was placed into an ice
bath, and the reaction was quenched by the addition of 15 mL of 10 N NaOH via addition funnel.
The still acidic mixture was neutralized with slow addition of saturated NaHCO3 to pH 8.0
(approx. 100 mL total volume). The mixture was extracted with CH2Cl2 (2 × 100 mL), and the
101
combined organic layers were then washed with NaHCO3 (150 mL) and brine (150 mL), then
dried over MgSO4 and filtered. The solvent was removed on a rotary evaporator, and the crude
product was purified via SiO2 flash column chromatography with an eluent of 25% to 35%
diethyl ether in pentane, and yielded 338 mg (77%) of the pure product as an off-white solid,
m.p. 132–134 °C. 1H NMR (400 MHz, CDCl3): δ 7.16 (t, J = 8.0 Hz, 1H), 6.95 (dd, J = 7.9, 1.0
Hz, 1H), 6.67 (dd, J = 8.1, 1.0 Hz, 1H), 3.76 (br s, 2H); 13
C NMR (151 MHz, CDCl3): δ 189.49,
141.17, 133.22, 127.60, 117.46, 113.51, 113.40. HRMS-ESI (m/z): [M+H]+ calcd. for
C7H6NOS2, 183.9885; found, 183.9874.
Analog 4.21. The procedure used for 4.20 was followed with 37 mg (0.20 mmol) of the
amine 4.19. The procedure yielded 14 mg (25% over two steps) of the product as an off-white
solid, m.p. 162–166 °C (dec). 1H NMR (600 MHz, approx. 15% CD3OD in DMSO-d6): δ 7.08–
7.03 (m, 1H), 6.97–6.94 (m, 1H), 6.83–6.79 (m, 1H); 13
C NMR (151 MHz, approx. 15% CD3OD
in DMSO-d6): δ 152.97, 144.97, 131.60, 123.47, 121.69, 118.22. HRMS-ESI (m/z): [M–Cl]+
calcd. for C6H6NS5, 251.9098; found, 251.9097.
Compound 4.23. 2-Chloro-1-nitro-4-(trifluoromethyl)benzene 4.22 (895 mg, 3.50 mmol)
was dissolved in 7 mL of DMSO and was treated with 627 mg (3.50 mmol) of sodium
dimethyldithiocarbamate dihydrate in a sealed vial in a microwave reactor at 200 °C for 20 min.
The mixture was diluted to 70 mL with water and extracted into 70 mL of CH2Cl2. The organic
layer was washed with brine (70 mL) and dried over MgSO4. The solids were removed by
filtration, and the volatile components were removed via rotary evaporation. The crude product
was purified by reversed-phase flash column chromatography with a gradient of 10% to 100%
acetonitrile in water (0.1% TFA). Fractions containing product were combined and the organic
portion of the solvent was evaporated. The aqueous phase was neutralized by addition of
saturated NaHCO3 and was then extracted with ethyl acetate. The organic layer was evaporated
yielding 336 mg (41%) of product 4.23 as a light brown oil, which was taken on directly to the next step.
1H NMR (400 MHz, CDCl3): δ 8.10 (d, J = 2.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.58
(dd, J = 8.0, 2.0 Hz, 1H).
Analog 4.24. The procedure for pentathiepin formation for 4.20 was followed with 330 mg
of 4.23, without the need for salt formation after the purification. The procedure yielded 63 mg
(15%) of the product as a yellow oil. 1H NMR (400 MHz, CDCl3): δ 8.14 (d, J = 2.0 Hz, 1H),
8.01 (d, J = 8.1 Hz, 1H), 7.61 (dd, J = 8.1, 2.0 Hz, 1H); 19
F NMR (376 MHz, CDCl3): δ –62.97; 13
C NMR (126 MHz, CDCl3): δ 148.13, 145.08, 136.49, 132.84 (q, JCF = 3.6 Hz), 132.37 (q, JCF
= 33.4 Hz), 127.04 (q, JCF = 3.7 Hz), 122.99 (q, JCF = 273.1 Hz). Anal. calcd. for C7H3F3S5: C,
102
27.62; H, 0.99; N, 0.00; S, 52.67; found: C, 27.16; H, 0.96; N, < 0.02; S, 50.80. The 1H NMR
spectrum correlates with the published data.17
Analog 4.27. In a reaction vial charged with a magnetic stir bar, the starting aniline 4.08 (127
mg, 0.400 mmol) was dissolved in 1.8 mL of ethanol, and the vial was placed in a 0 °C ice bath.
Acetaldehyde (2.0 mL of a 0.5 M solution in ethanol, 1.0 mmol, 2.5 equiv) was added via
syringe, followed by 0.2 mL of glacial acetic acid. This solution was stirred 30 min at 0 °C, at
which point, a suspension of 75 mg (1.2 mmol, 3.0 equiv) of NaCNBH3 in EtOH (0.5 mL) was
added to the reaction vial via syringe. The resulting reaction mixture was stirred at 0 °C for 1 h.
The reaction was quenched with the addition of 1.5 mL of concentrated HCl, and then the
mixture was neutralized to pH 8.0 with saturated NaHCO3. The resulting mixture was extracted
into CH2Cl2 (2 × 30 mL), and the combined organic layer was dried over MgSO4. The solids
were removed by filtration, and the volatile components were removed via rotary evaporation.
The resulting residue was purified by SiO2 flash column chromatography with 0% to 10%
EtOAc in hexanes as the eluent. Compound 4.27 was isolated as a yellow solid (15 mg, 11%), m.p. 119–124 °C (dec).
1H NMR (500 MHz, CDCl3): δ 7.24 (d, J = 1.7 Hz, 1H), 6.77 (d, J = 1.7
Hz, 1H), 5.37 (s, 1H), 3.24 (qd, J = 7.2, 5.0 Hz, 2H), 1.34 (t, J = 7.2 Hz, 3H); 19
F NMR (376 MHz, CDCl3): δ –63.64;
13C NMR (126 MHz, CDCl3): δ 151.36, 146.72, 133.34 (q, JCF = 32.3
Hz), 127.53, 123.22 (q, JCF = 273.0 Hz), 118.92 (q, JCF = 3.5 Hz), 108.99 (q, JCF = 4.3 Hz),
38.59, 14.49. HRMS-ESI (m/z): [M+H]+ calcd. for C9H9F3NS5, 347.9285; found, 347.9287.
Analog 4.28. Following the procedure above for 4.27, with a 0.5 M solution of benzaldehyde
in EtOH, the product was obtained as a yellow solid (12 mg, 7%), m.p. 97–104 °C (dec). 1H
NMR (500 MHz, CDCl3): δ 7.43–7.30 (m, 5H), 7.28 (d, J = 1.8 Hz, 1H), 6.80 (d, J = 1.8 Hz,
1H), 5.85 (br t, 1H), 4.43 (d, J = 5.5 Hz, 2H); 19
F NMR (376 MHz, CDCl3): δ –63.66; 13
C NMR
(126 MHz, CDCl3): δ 151.16, 146.77, 137.14, 133.32 (q, JCF = 32.8 Hz), 129.19, 128.10, 127.49,
123.10 (q, JCF = 273.7 Hz), 119.54 (q, JCF = 3.8 Hz), 109.45 (q, JCF = 3.7 Hz), 48.17. HRMS-ESI
(m/z): [M+H]+ calcd. for C14H11F3NS5, 409.9442; found, 409.9437.
Analog 4.29. Diamine 4.10 (5 mg) was dissolved in 0.5 mL of ether and treated with 4 drops
of concentrated HCl to yield 6 mg (quant) of 4.29 as an off-white solid, m.p. 152–155 °C (dec). 1H NMR (400 MHz, DMSO-d6): δ 6.97 (d, J = 1.9 Hz, 1H), 6.65 (d, J = 1.9 Hz, 1H);
19F NMR
103
(376 MHz, DMSO-d6): δ –63.30. HRMS-ESI (m/z): [M–2HCl+H]+ calcd. for C14H9F6N2S6,
510.8989; found, 510.8983.
General procedures for determination of inhibitor IC50
Reaction volumes of 100 μL were used in 96-well plates. 75 L of water was added to each
well, followed by 5 L of 20 × buffer (stock: 1 M imidazole-HCl, pH 7.0, 1 M NaCl, 0.2%
Triton-X 100). Five L of the appropriate inhibitor dilution in DMSO was added, followed by 5
L of STEP (stock: 0.2 M, 10 nM in assay). The assay plate was then incubated at 27 °C for 10
min with shaking. The reaction was started by addition of 10 L of 10 × pNPP substrate (stock: 5
mM, 500 M in assay), and reaction progress was immediately monitored at 405 nm at a
temperature of 27 °C. The initial rate data collected was used for determination of IC50 values.
For IC50 determination, kinetic values were obtained directly from nonlinear regression of
substrate-velocity curves in the presence of various concentrations of inhibitor using one site
competition in GraphPad Prism v5.01 scientific graphing software. The Km value of pNPP for
STEP under these conditions was determined to be 745 M, and was used in the kinetic analysis.
For experiments with catalase or superoxide dismutase (SOD), 10 L of the appropriate
enzyme stocks (catalase: 800 U/mL stock, 80 U/mL in assay; SOD: 1000 U/mL stock, 100 U/mL
in assay) were added prior to addition of the inhibitor and STEP.
For the experiments with glutathione reducing agent, 10 L of glutathione (stock: 10 mM, 1
mM in assay) was added before the inhibitor stocks, and only 65 L of water was added initially
to maintain the 100 L assay volume. Once the inhibitor stocks were added, the assay plate was
allowed to incubate for 10 min at 27 °C with shaking. This was followed by addition of STEP
(stock: 1.0 M, 50 nM in assay) and another 10 min incubation at 27 °C prior to addition of
pNPP substrate.
General procedures for determination of kinact/Ki
The second-order rate constants of inactivation were determined under pseudo-first order
conditions using the progress curve method.8 Assay wells contained a mixture of the inhibitor
(800, 400, 200, 100, 50, 0 nM) and 745 M of pNPP (Km = 745 M) in buffer (50 mM
imidazole-HCl pH 7.0, 50 mM NaCl, 0.01% Triton-X 100). Aliquots of STEP were added to
each well to initiate the assay. The final concentration of STEP was 10 nM. Hydrolysis of pNPP
was monitored spectrophotometrically for 30 min at an absorbance wavelength of 405 nm. To
determine the inhibition parameters, time points for which the control ([I] = 0) was linear were
used. A kobs was calculated for each inhibitor concentration via a nonlinear regression of the data
according to the equation P=(vi/kobs)(1–e^(–kobst)) (where P = product formation, vi = initial rate,
t = time (s)) using Prism v5.01 (GraphPad). Because kobs varied hyperbolically with [I], nonlinear
regression was performed to determine the second-order rate constant, kinact/Ki, using the
equation kobs=kinact[I]/([I]+Ki(1+[S]/Km)). Assays were performed in quadruplicate. The average
and standard deviation of the assays is reported.
104
Dialysis
STEP was diluted into 1 × assay buffer with either inhibitor or DMSO control (final volume:
2.9 mL, final concentration: 1 M STEP, 5 M TC-2153; 50 mM imidazole-HCl, pH 7.0, 50
mM NaCl, 0.001% Triton-X 100, 5% v/v DMSO). The samples were shaken at ambient
temperature for 1 h to inhibit STEP. Each sample was then transferred to a separate Thermo
Scientific Slide-A-Lyzer dialysis cassette with a 10,000 MW cut off and 0.5–3.0 mL sample
volume and was dialyzed into 1 L of 1 × assay buffer over 24 h in a 4 °C cold room. Aliquots of
approximately 100 L were removed from the dialysis cassette at 0, 4, and 24 h time points.
Protein concentration was determined by reading absorbance at 280 nm compared to a standard
curve for STEP. The samples were diluted to 100 nM in 100 L of 1 × assay buffer. The reaction
was started by addition of 10 L of 10 × pNPP substrate (stock: 20 mM, 1.81 mM in assay; total
assay volume: 110 L), and reaction progress was immediately monitored at 405 nm at a
temperature of 27 °C. The initial rate data collected was used to determine enzyme activity
standardized to the DMSO control zero time point.
Determination of TC-2153 stability in imidazole buffer
To monitor the stability of TC-2153 in the imidazole buffer, 20 L of 20 mM TC-2153 stock
in DMSO was added to an Eppendorf tube. The solution was diluted to 400 L (1 mM TC-2153
final concentration, 5% final DMSO) with either water or the pH 7.0 imidazole buffer. The tube
was allowed to incubate at ambient temperature with shaking for 1 h. The mixture was diluted
with 150 L of DMSO-d6 and transferred to an NMR tube containing a capillary of
trifluoroacetic acid as an external standard (–76.55 ppm). The stability of the compound in the
buffer was confirmed by observing no differences in the 19
F NMR spectra (Figure 4.14). As a
control for compound modification, the experiment was repeated with the addition of 1 mM
GSH in the incubation buffer.
Recovery of STEP activity by reducing agents
STEP was diluted to 200 nM in water and aliquots of this stock were mixed with DMSO (5%
by volume) or TC-2153 (5 M final concentration, 5% DMSO by volume) and incubated at
ambient temperature on a shaker for 10 min. Each sample was aliquoted out and 50 L was
transferred to wells of a 96-well microtiter plate containing 40 L of 2 × assay buffer with added
reductant (GSH or DTT, 1 mM final concentration) and shaken for 0, 15, 30, or 60 additional
minutes at ambient temperature. The reaction was started by addition of 10 L of 10 × pNPP
substrate (stock: 20 mM, 2 mM in assay; total assay volume: 100 L), and reaction progress was
immediately monitored at 405 nm at a temperature of 27 °C. The initial rate data collected was
used to determine enzyme activity standardized to the DMSO controls.
105
Figure 4.14. TC-2153 stability in (a) water control, (b) imidazole buffer and (c) 1 mM GSH.
Mass spectrometry
To explore the protein modification(s) of STEP upon TC-2153 inhibition, reduced and
nonreduced gel purified STEP (WT or C472S mutant) proteins were analyzed by high-resolution
tandem mass spectrometry. Briefly, purified STEP WT or C-S mutant proteins (10 g each) were
incubated with vehicle (1% DMSO) or TC-2153 (10 M in 1% DMSO) in assay buffer (50 mM
imidazole-HCl, pH 7.0) at ambient temperature (25 °C) for 30 min. Samples were resolved on
8% SDS-PAGE or non-denaturing PAGE and proteins were visualized by Coomassie Blue
staining. Gel bands were excised and kept at –80 °C until use. Excised gel bands corresponding
to the mutant and WT STEP with and without TC-2153 were in-gel trypsin digested under native
conditions (without reducing agent) overnight. Peptides were extracted from the digested
samples with 80% acetonitrile containing 0.1% TFA, and then dried under SpeedVac. Samples
were then reconstituted in minimum solution containing 0.1% TFA, and loaded onto a RP C18
nanoACQUITY UPLC column (1.7 m BEH130 C18, 75 m×250mm, with a 5 m Symmetry
C18 2G-V/M Trap [180 m×20mm]). Eluted peptides were directly infused into an Orbitrap
Elite LC MS/MS system running data dependent acquisition. Acquired data were processed
utilizing Progenesis LCMS software (Nonlinear Dynamics) and MASCOT Search engine with
user defined possible modification(s) search criteria.
Western blotting
Samples were prepared and resolved by SDS-PAGE, transferred to nitrocellulose membrane
and incubated with phospho-specific antibodies (anti-pY204/187
ERK1/2, anti-pY402
Pyk2, anti-
pY1472
GluN2B) or total protein antibodies (anti-ERK2, anti-Pyk2 and anti-NR2B) overnight at 4
106
°C. All antibodies used are listed in Table 4.4. Immunoreactivity was visualized using a
Chemiluminescent substrate kit (Pierce Biotechnology, Rockford, IL) and detected using a
G:BOX with the image program GeneSnap (Syngene, Cambridge, UK). All densitometric
quantifications were performed using the Genetools program.
Table 4.4. Primary and secondary antibodies used in this study
Antibody Format Immunogen Host Dilution Source
Anti-pTyr1472
GluN2B
Whole IgG,
unconjugated
Synthetic
phosphopeptide Rabbit 1:1000
Cell Signaling
Technology,
Danvers, MA
Anti-GluN2B Whole IgG,
unconjugated
C-terminus of
mouse GluN2B Rabbit 1:1000
Cell Signaling
Technology
Anti-pTyr402
Pyk2
Whole IgG,
unconjugated
Synthetic
phosphopeptide of
human Pyk2
Rabbit 1:1000 Invitrogen
Anti-Pyk2 IgG2a C-terminus of
human Pyk2 Mouse 1:1000
Cell Signaling
Technology
Anti-pTyr204
ERK1/2
Whole IgG,
unconjugated
Synthetic
phosphopeptide Mouse 1:500
Santa Cruz
Biotechnology
Anti-ERK2 Whole IgG,
unconjugated
C-terminus of rat
p44 MAP Kinase Rabbit 1:20,000
Cell Signaling
Technology
Anti-GAPDH,
clone 6c5
IgG1,
unconjugated
Purified protein
from rabbit muscle Mouse 1:20,000 Millipore
Anti-rabbit
Whole IgG
peroxidase-
conjugated
Rabbit Fc Donkey 1:10,000 Amersham
Biosciences
Anti-mouse
Whole IgG
peroxidase-
conjugated
Mouse Fc Sheep 1:5,000 Amersham
Biosciences
In vivo assays
Wild-type male C57BL/6 mice (3–6 months) were used for all studies. An initial dose-
response curve was carried out using TC-2153 (1, 3, 6 and 10 mg/kg, i.p.). Pilot studies were
conducted to optimize the time after i.p. injection when STEP substrates showed maximum Tyr
phosphorylation (1–3 h). Cortical tissues were dissected out 3 h post injection and processed for
107
subcellular fractionation. Brain tissue was homogenized in TEVP buffer containing 10 mM Tris-
HCl, pH 7.6, 320 mM sucrose, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 20 mM NaF, 1 mM
Na3VO4, and protease inhibitors. Homogenates were centrifuged at 800 × g to remove nuclei and
large debris (P1). Synaptosomal fractions (P2) were prepared from S1 by centrifugation at 9,200
× g for 15 min. The P2 pellet was washed twice and was resuspended in TEVP buffer. In some
experiments, mice were injected with TC-2153 (3 mg/kg, i.p.) and cortex, cerebellum and spleen
were removed to test for the in vivo inhibition of the highly related PTPs, HePTP and PTP-SL.14
Behavioral analysis
A previous study showed that genetic reduction of STEP significantly reversed cognitive
deficits in 6-month old 3xTg-AD mice.1a
Here we were interested in testing whether
pharmacologic inhibition of STEP with TC-2153 had a similar beneficial effect in this AD
mouse model. We also wanted to test whether TC-2153 had any effects on cognition in WT
mice. The WT cohort was treated as an independent group, and analyses were made
independently for the WT and 3xTg-AD groups. WT or 3xTg-AD mice were randomly allocated
to treatment with either vehicle or TC-2153.
Morris water maze
The reference memory version of the MWM task was performed as described previously.18
A
crossover design was not used in the Morris water maze task, as the mice were randomly
assigned to each treatment condition and can be exposed to the task only once. Briefly, animals
were trained to swim in a 1.4 m diameter pool to find a submerged platform (14 cm in diameter)
located 1 cm below the surface of water (24 °C), rendered opaque by the addition of non-toxic
white paint. Animals were pseudo-randomly started from a different position at each trial and
used distal visual-spatial cues to find the hidden escape platform that remained in the center of
the same quadrant throughout all training days. Training measures included escape latency to
reach the platform, swim speed, and thigmotaxis. When animals failed to find the platform they
were guided to it and remained there for 10 s before removal. 24 h after the acquisition phase, the
platform was removed and a probe trial of 90 s was given to evaluate the number of entries in a
circular zone (three times the platform diameter) positioned around the previous platform
location (target zone) and in the opposite quadrants. To assess visual deficits and motivation to
escape from water, the probe test was followed by a cued task (60 s; three trials per animal)
during which the platform was visible. The visible platform was moved to different locations
between each trial. After each trial, animals were immediately placed under a warming lamp to
dry to prevent hypothermia. The experimenter was blind to mouse genotype when administering
TC-2153 or vehicle to AD mice (AD-TC, n =7; AD-Veh, n = 6) or WT mice (WT-TC, n = 13;
WT-Veh, n= 12). Behavioral data from training, probe, and cued trials were acquired and
analyzed using the ANY-maze automated tracking system (Stoelting, IL, USA).
Data analysis
For the Morris water maze training and probe sessions, a 3-way repeated measures analysis
of variance (ANOVA) with 2 between-subject (Genotype, Treatment) and 1 within-subject
(Training day or Quadrant) factor was used. Escape latency (training) and number of entries
108
(probe) were the dependent measures (StatView, Cary, NC). Swim speed and escape latency
during the probe and cued trials, respectively were analyzed using a 2-way ANOVA with
Genotype and Treatment as the between subject factors. Post-hoc analyses were conducted on
significant results. For cell-based assays, one-way ANOVA with post hoc Bonferroni test was
used to determine statistical significance. All data were expressed as mean ± s.e.m.
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110
111
Chapter 5. Seleninic acids as redox-reversible inhibitors of STEP
Abstract: This chapter outlines the use of seleninic acids as redox-reversible inhibitors of STEP.
The redox-reversible mode of inhibition described in chapter 4 is adapted to seleninic acids,
which have been demonstrated to form stable S–Se bonds with cysteine thiols. This new PTP
pharmacophore is merged with the SAR determined in chapter 3 to attain an inhibitor with good
activity in vitro. This work is unpublished.
112
Authorship
I carried out all of the work described in this chapter. I synthesized all of the compounds and
determined their in vitro inhibition against STEP.
Introduction
Selenium is an essential micronutrient of many living species, including humans, because the
selenocysteine amino acid is necessary for the activity of a few important antioxidant enzymes.
In high doses, however, selenium can be toxic. In 2007, Plateau and coworkers were able to
account for some of this toxicity by studying its effects in Saccharomyces cerevisiae.1 Selenite
(SeO32–
, 5.01) reacts with readily with excess reduced glutathione (GSH) to generate hydrogen
selenide (HSe–, 5.04), which is toxic in S. cerevisiae (Scheme 5.1). Other reduction products,
such as selenodiglutathione 5.02, reactive oxygen species (ROS) or elemental Se0, were found to
not be toxic. These results demonstrate that selenium can undergo multiple redox reactions with
bio-relevant thiols.
Scheme 5.1. Selenium redox in vivo with GSH
In 2008, Zhang and coworkers reported the first use of a seleninic acid as a phosphatase
inhibitor.2 They synthesized tyrosine derived seleninic acid 5.06 and characterized its irreversible
inhibition consistent with forming a selenium–sulfur bond at the active site cysteine residue of
both YopH and PTP1B. Seleninic acid 5.06 had a kinact/Ki of 91 ± 20 M–1
s–1
against YopH and
only 14 ± 10 M–1
s–1
against PTP1B. Additionally, they demonstrated that seleninic acid 5.06
forms a covalent bond with cysteine derivative 5.07 and were able to characterize selenosulfide
adduct 5.08 (Scheme 5.2). Finally, to directly demonstrate the irreversible adduct, they obtained
a crystal structure showing a covalent bond between compound 5.06 and the active site of
PTP1B.
Scheme 5.2. Seleninic acid 5.6 forms a covalent adduct with cysteine derivative 5.7
Another important example of selenium redox in vivo is the catalytic mechanism of
thioredoxin reductase (TrxR) (Figure 5.1).3 As the only known enzyme to catalyze the reduction
of thioredoxin (Trx),4 it is a central component of the Trx system which exists in all living cells
and is important for maintaining the cellular redox environment.5 In one of the active sites of
TrxR is a selenocysteine residue which forms a selenosulfide with a neighboring cysteine upon
113
oxidation by Trx. The selenosulfide is reduced to the free selenol and thiol through a series of
reactions that utilize NADPH as the terminal reductant.
Figure 5.1. Selenosulfide redox cycling in Trx/TrxR system.
Combining the facts that selenosulfides have been formed from seleninic acids with active
site cysteines in PTP active sites and that they possess reversible redox in vivo as exemplified by
the TrxR cycle, we hoped that we could obtain redox-reversible inhibitors by introducing the
seleninic acid functional group as a pharmacophore. This pharmacophore should react in in vitro
assays similarly to the pentathiepins in chapter 4, and because it can be incorporated into
druglike scaffolds, it should enable more straightforward modulation of physicochemical
properties.
Synthesis of seleninic acid inhibitors
Utilizing the procedures of Zhang, et. al., we first synthesized seleninic acid 5.13 (Scheme
5.3).2 First, (2-phenyl)-selenoacetic acid 5.10 is generated by treatment of phenylacetic acid with
Woollins’ reagent (the Se analog of Lawesson's reagent). The selenoacetic acid then functions as
the nucleophile in a Mitsunobu reaction yielding the selenoester 5.12. Treatment with
dimethyldioxirane (DMDO)6 yields the desired seleninic acid 5.13.
Scheme 5.3. Synthesis of seleninic acid inhibitor 5.13
With the simple biphenyl inhibitor synthesized, we next sought to incorporate the SAR
optimized scaffold of 3.97 and 3.98 from chapter 3 (Scheme 5.4). Lithiation of 1-bromo-3-
iodobenzene followed by addition into 3,4-dichlorobenzaldehyde yielded aryl bromide 5.15.
114
Subsequent Miyaura borylation7 gave boronate ester 5.16. Protection of the benzyl alcohol as the
TBS ether followed by Suzuki-Miyaura cross-coupling reaction with 4-bromobenzyl alcohol
afforded benzyl alcohol 5.18. The Mitsunobu reaction and DMDO oxidation afforded seleninic
acid 5.20, which was deprotected with TBAF to afford the final seleninic acid inhibitor, 5.21.
Scheme 5.4. Synthesis of seleninic inhibitor 5.21
In chapter 3 we found that the -hydroxyphosphonic acid was more potent than the simple
methylene phosphonic acid. However, primarily out of concern for compound stability, the
literature precedented methylene seleninic acids were synthesized to test the viability of the
seleninic acid as an inhibitor. In this study, we desired to test the ability of the seleninic acid to
function as a redox-reversible pharmacophore for PTP inhibition without the added complication
of potential compound instability.
In vitro evaluation of inhibitors
As was the case in chapter 4 with the pentathiepins, we thought it was important to test the
activity of seleninic acids 5.13 and 5.21 both with and without the ubiquitous biological reducing
agent GSH (Table 5.1).8 Gratifyingly, the compound with the SAR optimized scaffold, 5.21, was
more potent than the simple biphenyl derivative, 5.13. Additionally, compound 5.21 was as
potent as TC-2153 in the absence of GSH. Interestingly, when GSH was added to the enzyme
assay, the potency of 5.21 was not affected nearly as much as that of TC-2153, maintaining sub-
M potency. This suggests that either the seleninic acid pharmacophore in 5.21 is not as
115
susceptible to the non-productive reaction with GSH, or that it forms a single stable adduct that
is still able to inhibit STEP at a reduced potency.
Table 5.1. IC50 values of seleninic acids against STEP compared to TC-2153
compound IC50 (nM)a,b
IC50 (M) a,c
seleninic acid 5.13 70 ± 10 > 50
seleninic acid 5.21 27 ± 7 0.53 ± 0.02
pentathiepin TC-2153 24.6 ± 0.8d
8.8 ± 0.4d
aAssays were performed in duplicate (mean ± S.D.).
bAssays contained no
GSH. cAssays contained 1 mM GSH.
dAssays were performed in
quadruplicate (mean ± S.D.)
The second-order rate of inactivation for each of the seleninic acid inhibitors was then
determined using the progress curve method (Figure 5.2).9 A kobs was determined for pNPP in
the presence of varying initial inhibitor concentrations (n = 4). Values were then analyzed with
non-linear regression to obtain the kinetic constants: 5.13: kinact = 0.0170 ± 0.0029 s–1
; Ki = 1,011
± 300 nM; kinact/Ki = 16,800 ± 5,700 M–1
s–1
; 5.21: kinact = 0.0141 ± 0.0009 s–1
; Ki = 367 ± 63 nM;
kinact/Ki = 38,400 ± 7,000 M–1
s–1
. Consistent with the results in the IC50 assays and with the
elaborated scaffold providing an increase in binding, the Ki of 5.21 was approximately three
times more potent than the simple biphenyl scaffold, while their kinact values were the same
(within experimental error). For comparison, the only other seleninic acid PTP inhibitor reported
to date, 5.06, had a kinact/Ki of 91 ± 18 M–1
s–1
against YopH, considerably worse than the activity
of inhibitor 5.21 against STEP.
Figure 5.2. The progress curve method was used to determine the second-order rates of inactivation for
(a) inhibitor 5.13 (kinact/Ki = 16,800 ± 5,700 M–1
s–1
) and (b) inhibitor 5.21 (kinact/Ki = 38,400 ± 7,000
M–1
s–1
).
Conclusions
Seleninic acids have been shown to be redox-reversible inhibitors of STEP. Like the
pentathiepins in chapter 4, inhibition is dependent on the presence of GSH in the assays.
Moreover, the seleninic acid does not appear to be as affected by GSH, indicating that it perhaps
is not as reactive as the pentathiepin, or that it forms a stable adduct with GSH that can also
function as an inhibitor. Currently, inhibitors 5.13 and 5.21 have been submitted to our
collaborators in Dr. Paul Lombroso’s group to test for activity in neuronal cultures. Additionally,
116
researchers at AstraZeneca are working on obtaining crystal structures of the inhibitors in
complex with STEP. Through personal correspondence, they have been interested in obtaining
STEP inhibitors for the last two years but have not been able to generate anything as potent as
the inhibitors that we have disclosed in chapters 3, 4 and 5.
Experimental
General synthetic methods
Unless otherwise noted, all reagents were obtained from commercial suppliers and used
without further purification. Tetrahydrofuran (THF), dioxane, CH2Cl2, and diethyl ether (Et2O)
were passed through a column of activated alumina (type A2, 12 × 32, Purify Co.) under
nitrogen pressure immediately prior to use. All 1H,
13C, and
77Se NMR spectra were obtained at
ambient temperature on a Bruker AVB-400, AVB-500 or AVB-600 spectrometer. NMR
chemical shifts are reported in ppm relative to TMS (0.00), CHCl3 (7.26), or CH3OH (3.31) for 1H, CDCl3 (77.16), or CD3OD (49.00) for
13C, and PhSeSePh (460.0) for
77Se. Mass
spectrometry (HRMS, ESI) are reported in m/z. Chromatography was performed either with
SiliCycle SiliaFlash P60 230−400 mesh silica gel or by utilizing a Biotage SP1 flash purification
system (Biotage model SP1-B1A). Reversed-phase purifications were conducted with a
Teledyne Isco CombiFlash Rf system equipped with HP C18 gold cartridges. Syringe filtrations
were performed with Millex-HN 0.45 µm Nylon 33 mm syringe filters. Product yields are not
optimized. Enzymatic assays were carried out on a BioTek Synergy 2 multimode microplate
reader.
Synthesis of inhibitors 5.13 and 5.21
Compound 5.12. Selenoester 5.12 was prepared by modifying a literature procedure for a
related compound.2 A solution of 1.10 g (4.20 mmol) of triphenylphosphine in 10 mL of THF
was stirred at 0 °C. Diisopropyl azodicarboxylate (94%, 904 mg, 4.20 mmol) was added
dropwise, and the reaction mixture was maintained at 0 °C for 5 min until the white
phsophonium intermediate was formed. A solution of 4-phenylbenzyl alcohol 5.11 (387 mg, 2.10
mmol) in 10 mL of THF was added dropwise by syringe. After 5 min of stirring, 20 mL of a
toluene solution of (2-phenyl)-selenoacetic acid 5.10 was added dropwise by syringe through a
syringe filter [5.10 was prepared by heating at 105 °C a 20 mL of toluene solution of 863 mg
(6.33 mmol) of phenylacetic acid and 1.00 g (1.90 mmol) of Woollins’ reagent for 1 h]. The
reaction mixture was allowed to warm to ambient temperature and then was stirred for 3 h. The
solution was concentrated under reduced pressure. Purification by flash chromatography on SiO2
with 95:5 hexanes/ethyl acetate yielded 230 mg (30%) of pure 5.12 as a pink solid, m.p. 83–86 °C.
1H NMR (500 MHz, CDCl3): δ 7.57 (d, J = 7.5 Hz, 2H), 7.50 (d, J = 8.1 Hz, 2H), 7.44 (app t,
117
J = 7.7 Hz, 2H), 7.40–7.28 (m, 8H), 4.18 (s, 2H), 3.89 (s, 2H); 13
C NMR (151 MHz, CDCl3): δ
200.36, 140.82, 139.98, 138.09, 132.87, 130.19, 129.44, 128.86, 128.84, 127.91, 127.43, 127.37,
127.12, 54.02, 29.20; 77
Se NMR (95 MHz, CDCl3): δ 635.17. MS-ESI (m/z): [M+H]+ calcd. for
C21H19O[80]
Se, 367.06; found, 367.2.
Inhibitor 5.13. Seleninic acid 5.13 was prepared following a literature procedure for a
related compound.2 Dimethyldioxirane (DMDO) was added to a stirred solution of 5.12 (170 mg,
0.47 mmol) in 5 mL of acetone until all of 5.12 was consumed according to LCMS analysis
(total ~10 mL of a 0.16 M solution of DMDO in acetone, 3.5 equiv). The reaction mixture was
concentrated, and the crude residue was purified via reversed-phase gradient column
chromatography (5−100% acetonitrile in water with 0.1% trifluoroacetic acid buffer) to yield
seleninic acid 5.13 as a white crystalline solid, m.p. 118–119 °C. 1H NMR (500 MHz, CD3OD):
δ 7.63 (m, 4H), 7.51–7.39 (m, 4H), 7.34 (t, J = 7.4 Hz, 1H), 4.29, 4.25 (ABq, J = 12.0 Hz, 2H); 13
C NMR (126 MHz, CD3OD): δ 142.58, 141.71, 132.17, 129.93, 129.42, 128.62, 128.33,
127.95, 62.14; 77
Se NMR (96 MHz, CD3OD): δ 1291.68. HRMS-ESI (m/z): [M–H]– calcd. for
C13H11O2[80]
Se, 278.9930; found, 279.0068 (Figure 5.3).
Figure 5.3. HRMS of 5.13. Major contributors to the main peaks are labeled with stable Se isotopes.
Natural abundances of Se isotopes: 74
Se, 0.87%; 76
Se, 9.36%; 77
Se, 7.63%; 78
Se, 23.78%; 80
Se, 49.61%; 82
Se, 8.73%.
118
Compound 5.15. The synthesis of compound 5.15 is described in chapter 3. It is identified in
chapter 3 as compound 3.105. 1H NMR provided here for reference.
1H NMR (400 MHz,
CDCl3): δ 7.51 (m, 1H), 7.48 (m, 1H), 7.44−7.40 (m, 2H), 7.27−7.16 (m, 3H), 5.74 (d, J = 3.2
Hz, 1H), 2.31 (d, J = 3.2 Hz, 1H).
Compound 5.16. Following a literature procedure,
10 a 250 mL flame-dried round bottom
flask was charged with a magnetic stir bar, crude 5.15 (6.65 g, 20.0 mmol),
bis(pinacolato)diboron (10.16 g, 40.0 mmol), potassium acetate (5.89 g, 60.0 mmol) and DMSO
(80 mL). The solution was sparged with N2 at ambient temperature with stirring for 30 min.
Pd(dppf)Cl2 (816 mg, 1.00 mmol, 5 mol %) was added to the solution, which was sparged with
N2 for an additional 30 min. The reaction flask was fitted with a rubber septum and placed in a
pre-heated 80 °C oil bath, and the reaction mixture was stirred under N2 for 6 h. The reaction
flask was removed from the oil bath, and the mixture was allowed to cool to ambient
temperature. The reaction mixture was diluted with 700 mL of water and extracted into Et2O (3 ×
700 mL). The combined organic layer was washed with brine (1 L) and the volatiles were
removed under reduced pressure. The crude residue was purified by silica gel chromatography
with an eluent of 4:1 hexanes:ethyl acetate, yielding 5.16 as a colorless oil (3.34 g, 44% over 2 steps).
1H NMR (400 MHz, CDCl3): δ 7.80–7.79 (m, 1H), 7.75 (d, J = 7.2 Hz, 1H), 7.51 (d, J =
2.0 Hz, 1H), 7.44–7.31 (m, 3H), 7.20 (dd, J = 8.4, 2.1 Hz, 1H), 5.79 (s, 1H), 2.04 (s, 1H), 1.34 (s, 12H);
13C NMR (151 MHz, CDCl3): δ 144.03, 142.40, 134.82, 132.93, 132.61, 131.41, 130.49,
129.53, 128.54, 128.45, 126.00, 84.13, 75.32, 25.02, 25.00.NMR spectral data of racemic 5.16
was identical to literature spectra of each of the enantioenriched isomers.10
Compound 5.17. A 250 mL flame-dried round bottom flask was charged with a magnetic
stir bar, 5.16 (4.55 g, 12.0 mmol), imidazole (6.54 g, 96.0 mmol), and TBSCl (7.23 g, 48.0
mmol), and the flask was flushed with N2. DMF (15 mL) was added, and the reaction mixture
was stirred at ambient temperature with monitoring by TLC. After 1 h, the reaction was stopped
by addition of 20 mL of saturated aqueous NaHCO3, and the mixture was diluted with 200 mL of
water. The resulting suspension was extracted with CH2Cl2 (4 × 150 mL), and the combined
organic layer was washed with water (400 mL) and brine (400 mL), dried over MgSO4, filtered
and the volatiles were removed under reduced pressure. The crude residue was purified by silica
gel chromatography with an eluent of 9:1 hexanes:ethyl acetate, yielding 5.17 as a white solid
119
(4.18 g, 71%); m.p. 102–105 °C. 1H NMR (500 MHz, CDCl3): δ 7.75–7.67 (m, 2H), 7.50–7.47
(m, 2H), 7.37–7.31 (m, 2H), 7.21 (dd, J = 8.3, 2.0 Hz, 1H), 5.72 (s, 1H), 1.35 (s, 12H), 0.92 (s, 9H), 0.02 (s, 3H), –0.05 (s, 3H);
13C NMR (126 MHz, CDCl3): δ 145.80, 143.48, 134.21, 132.62,
132.30, 130.80, 130.31, 129.16, 128.19, 128.17, 125.70, 83.96, 75.79, 25.94, 25.06, 24.98, 18.41,
-4.63, -4.74.
Compound 5.18. A 250 mL round bottom flask was charged with a magnetic stir bar, 5.17
(4.08 g, 8.27 mmol), 4-bromobenzyl alcohol (1.55 g, 8.27 mmol), sodium carbonate (5.26 g, 49.6
mmol), Pd(PPh3)4 (485 mg, 0.420 mmol) and 42 mL of a solvent mixture of 4:1:1
DME:EtOH:H2O. The reaction mixture was sparged with N2 at ambient temperature with stirring
for 45 min. The reaction flask was fitted with a rubber septum and placed in a pre-heated 85 °C
oil bath, and the reaction mixture was stirred under N2 for 20 h. The reaction mixture was
allowed to cool to ambient temperature then was filtered through a pad of Celite; the filter cake
was washed with EtOAc. Volatiles were removed under reduced pressure, and the resulting
crude residue was purified by silica gel chromatography with an eluent of 4:1 hexanes:ethyl
acetate, yielding 5.18 as a yellow oil (3.15 g, 77 %). 1H NMR (500 MHz, CDCl3): δ 7.61–7.55
(m, 3H), 7.53 (s, 1H), 7.51–7.43 (m, 3H), 7.42–7.36 (m, 2H), 7.32 (d, J = 7.7 Hz, 1H), 7.29–7.20
(m, 1H), 5.78 (s, 1H), 4.75 (s, 2H), 1.98 (s, 1H), 0.96 (s, 9H), 0.06 (s, 3H), 0.03 (s, 3H); 13
C
NMR (126 MHz, CDCl3): 145.60, 144.79, 141.10, 140.50, 140.14, 132.40, 131.00, 130.39,
129.03, 128.21, 127.58, 127.41, 126.37, 125.66, 125.33, 124.95, 75.77, 65.13, 25.93, 18.41,
–4.62, –4.69. HRMS-ESI (m/z): [M+H–H2O]+ calcd. for C26H29
[35]Cl2OSi, 455.1359; found,
455.1356.
Compound 5.19. Compound 5.19 was prepared following the procedure for 5.12 beginning
with 2.00 g (4.20 mmol) of 5.18. The procedure yielded 650 mg (24%) of 5.19 as an orange oil. 1H NMR (400 MHz, CDCl3): δ 7.51 (br t, 1H), 7.49 (d, J = 1.9 Hz, 1H), 7.48–7.39 (m, 3H),
7.40–7.23 (m, 10H), 7.21 (dd, J = 8.4, 2.0 Hz, 1H), 5.73 (s, 1H), 4.15 (s, 2H), 3.88 (s, 2H), 0.94 (s, 9H), 0.03 (s, 3H), –0.00 (s, 3H);
13C NMR (151 MHz, CDCl3): δ 200.36, 145.61, 144.77,
141.01, 139.77, 138.32, 132.87, 132.41, 130.99, 130.40, 130.21, 129.49, 129.01, 128.85, 128.20,
127.93, 127.43, 126.30, 125.65, 125.29, 124.91, 75.79, 54.04, 29.18, 25.95, 18.43, –4.60, –4.69;
120
77Se NMR (95 MHz, CDCl3): 635.94. MS-ESI (m/z): [M+H–C6H5COSeH]
+ calcd. for
C26H29[35]
Cl2OSi, 455.14; found, 455.1.
Compound 5.20. Compound 5.20 was prepared following the procedure for 5.13 beginning
with 630 mg (0.96 mmol) of 5.19. The reaction required a total of ~24 mL of a 0.16 M solution
of DMDO in acetone (4.0 equiv) for all of 5.19 to be consumed (monitored by LCMS). The
crude residue was purified first by reversed-phase gradient column chromatography (20−100%
acetonitrile in water with 0.1% trifluoroacetic acid buffer), then by flash chromatography on
SiO2 with an eluent of 94:5:1 CH2Cl2/MeOH/formic acid to yield 5.20 (180 mg, 33%) as a red
viscous oil. The product was contaminated with ~5–10% of an unknown impurity that was co-
purified after both regular and reversed-phase purification. 1H NMR is provided for the desired
product. 1H NMR (400 MHz, CDCl3): δ 7.59–7.27 (m, 10H), 7.20 (dd, J = 8.3, 2.0 Hz, 1H), 5.73
(s, 1H), 4.43 (s, 2H), 0.92 (s, 9H), 0.02 (s, 3H), –0.01 (s, 3H). HRMS-ESI (m/z): [M+H–
Se(OH)2]+ calcd. for C26H29
[35]Cl2OSi, 455.1359; found, 455.1374.
Inhibitor 5.21. To a stirred solution of 5.20 (169 mg, 0.288 mmol) in THF (3 mL) at ambient
temperature was added tetra-n-butylammonium fluoride (1.0 M solution in THF, 0.29 mL, 0.29
mmol, 1 equiv) dropwise via syringe. After 30 min all of 5.20 was consumed (monitoring by
LCMS), and the reaction was concentrated. The crude residue was purified by reversed-phase
gradient column chromatography (10−100% acetonitrile in water with 0.1% formic acid buffer)
to yield 5.21 (40 mg, 31%) as a white solid, m.p. 95–99 °C. 1H NMR (500 MHz, CD3OD): δ
7.64–7.55 (m, 4H), 7.51 (d, J = 7.7 Hz, 1H), 7.47–7.37 (m, 4H), 7.32 (d, J = 7.7 Hz, 1H), 7.29
(d, J = 8.5 Hz, 1H), 5.81 (s, 1H), 4.27, 4.23 (ABq, J = 12.0 Hz, 2H); 13
C NMR (151 MHz, CD3OD): δ 146.97, 145.88, 142.31, 141.97, 133.16, 132.20, 131.82, 131.43, 130.16, 129.54,
128.35, 127.52, 127.23, 126.96, 126.19, 75.51, 62.12; 77
Se NMR (95 MHz, CD3OD): δ 1291.16.
HRMS-ESI (m/z): [M–H]– calcd. for C20H15
[35]Cl2O3
[80]Se, 452.9569; found, 452.9601 (Figure
5.4).
121
Figure 5.4. HRMS of 5.21. Major contributors to the main peaks are labeled with stable Se and Cl
isotopes. Natural abundances of Se isotopes: 74
Se, 0.87%; 76
Se, 9.36%; 77
Se, 7.63%; 78
Se, 23.78%; 80
Se,
49.61%; 82
Se, 8.73%. Natural abundance of Cl isotopes: 35
Cl, 75.77%; 37
Cl, 24.23%.
General procedures for determination of inhibitor IC50
Reaction volumes of 100 μL were used in 96-well plates. 75 L of water was added to each
well, followed by 5 L of 20 × buffer (stock: 1 M imidazole-HCl, pH 7.0, 1 M NaCl, 0.2%
Triton-X 100). Five L of the appropriate inhibitor dilution in DMSO was added, followed by 5
L of STEP (stock: 0.2 M, 10 nM in assay). The assay plate was then incubated at 27 °C for 10
min with shaking. The reaction was started by addition of 10 L of 10 × pNPP substrate (stock: 5
mM, 500 M in assay), and reaction progress was immediately monitored at 405 nm at a
temperature of 27 °C. The initial rate data collected was used for determination of IC50 values.
For IC50 determination, kinetic values were obtained directly from nonlinear regression of
substrate-velocity curves in the presence of various concentrations of inhibitor using one site
competition in GraphPad Prism v5.01 scientific graphing software. The Km value of pNPP for
STEP under these conditions was determined to be 745 M, and this value was used in the
kinetic analysis.
For the experiments with glutathione reducing agent, 10 L of glutathione (stock: 10 mM, 1
mM in assay) was added before the inhibitor stocks, and only 65 L of water was added initially
to maintain the 100 L assay volume. Once the inhibitor stocks were added, the assay plate was
allowed to incubate for 10 min at 27 °C with shaking. This was followed by addition of STEP
(stock: 1.0 M, 50 nM in assay) and another 10 min incubation at 27 °C prior to addition of
pNPP substrate.
General procedures for determination of kinact/Ki
The second-order rate constants of inactivation were determined under pseudo-first order
conditions using the progress curve method.9 Assay wells contained a mixture of the inhibitor
(2000, 666.7, 222.2, 74.1, 24.7, 0 nM) and 745 M of pNPP (Km = 745 M) in buffer (50 mM
imidazole-HCl pH 7.0, 50 mM NaCl, 0.01% Triton-X 100). Aliquots of STEP were added to
each well to initiate the assay. The final concentration of STEP was 10 nM. Hydrolysis of pNPP
122
was monitored spectrophotometrically for 30 min at an absorbance wavelength of 405 nm. To
determine the inhibition parameters, time points for which the control ([I] = 0) was linear were
used. A kobs was calculated for each inhibitor concentration via a nonlinear regression of the data
according to the equation P=(vi/kobs)(1–e^(–kobst)) (where P = product formation, vi = initial rate,
t = time (s)) using Prism v5.01 (GraphPad). Because kobs varied hyperbolically with [I], nonlinear
regression was performed to determine the second-order rate constant, kinact/Ki, using the
equation kobs=kinact[I]/([I]+Ki(1+[S]/Km)). Assays were performed in quadruplicate. The average
and standard deviation of the assays is reported.
References
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Plateau, P. J. Biol. Chem. 2007, 282, 8759.
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6. Crandall, J. K.; Curci, R.; D'Accolti, L; Fusco, C. e-EROS Encyclopedia of Reagents for
Organic Synthesis 2005.
7. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60, 7508.
8. Meister, A.; Anderson, M. E. Annu. Rev. Biochem. 1983, 52, 711.
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123
Chapter 6. Asymmetric additions of Knochel-type benzyl zinc reagents to N-tert-
butanesulfinyl aldimines
Abstract: Early in my graduate career, I wanted to be exposed to more of the chemistry in the
Ellman group than just the chemical biology aspects. There was a need in a project area that I
thought would provide me with valuable experience in organic synthesis. This chapter details the
development of a reaction for the addition of Knochel-type benzyl zinc reagents to N-tert-
butanesulfinyl aldimines. Notably, additions of these sp3-hybridized reagents demonstrate good
functional-group compatibility, adding chemoselectively to imines in the presence of esters and
nitriles under convenient ambient temperature conditions. Moreover, addition to a
glyceraldehyde-derived N-tert-butanesulfinyl imine proceeds in high yield and with exceptional
selectivity to provide rapid entry to hydroxyethylamine-based aspartyl protease inhibitors. The
majority of this work has been published (Buesking, A. W.; Baguley, T. D.; Ellman, J. A. Org.
Lett. 2011, 13, 964).
124
Authorship
The majority of the work described in this chapter was completed in collaboration with Dr.
Andrew Buesking, a fellow graduate student. Dr. Buesking and I both were involved in the
synthesis of the starting materials and the reaction optimization. Dr. Chris Incarvito (Yale
Chemical and Biophysical Instrumentation Center) solved the crystal structures and provided the
crystal data reports for products 6.27 and 6.31.
Introduction
The addition of nucleophiles to N-tert-butanesulfinyl imines is one of the most extensively
used approaches for the asymmetric synthesis of amines (Scheme 6.1).1 The popularity of these
methods results from several key features of the N-tert-butanesulfinyl imine substrates and their
corresponding sulfinyl-protected amine products. First, N-tert-butanesulfinyl imines (6.02) are
readily prepared in a single step, typically in high yield, from aldehydes and ketones with diverse
steric and electronic properties. Second, the imines are activated to nucleophilic addition, which
often proceeds with high diastereoselectivity. Third, imines 6.02 show good stability to
hydrolysis and tautomerization. Finally, after reaction with a nucleophile the N-tert-
butanesulfinyl amines 6.03 can undergo a mild deprotection to the final enantiomerically
enriched amine products (6.04).
Scheme 6.1. Asymmetric synthesis of amines via N-tert-butanesulfinamide chemistry
Grignard and organolithium reagents were the first nucleophiles to be used in this synthetic
process and proceed with high yields and diastereoselectivities for a broad range of coupling
partners.2 Still, these methods suffer from poor functional group compatibility, the need for low
reaction temperatures and limited commercial availability of the organometallic reagents, a
liability for analog synthesis. Rhodium-catalyzed additions of boron reagents to N-tert-
butanesulfinyl aldimines greatly expanded the breadth of functionality that may be present
during the addition step.3 However, these methods are currently limited to the coupling of aryl
and vinyl boron reagents, which are sp2-hybridized. Therefore, additions of sp
3-hybridized
organometallic reagents that also proceed with broad functional group compatibility would
represent a significant advance, but prior to our work, only functional group tolerant additions of
allylzinc4 and allylindium reagents
5 had primarily been reported.
Due to their high functional group compatibility, organozinc reagents have become
increasingly popular, particularly in Negishi cross coupling.6 While typical organozinc halides
(i.e., RZnX) do not react directly with aldehydes, ketones, or imines, Knochel and coworkers
have reported that benzyl zinc reagents prepared using a mixture of Mg0, LiCl, and ZnCl2 add
efficiently to aldehydes and ketones in high yields at ambient temperature (Scheme 6.1).7
Additionally, these sp3-hybridized organozinc reagents (6.06 or 6.07) demonstrate good
functional-group compatibility, tolerating both ester and nitrile groups. Because of these features,
125
we desired to apply these Knochel-type organozinc reagents in diastereoselective additions to N-
tert-butanesulfinyl aldimines.89
Scheme 6.2. Preparation of benzyl zinc reagents 6.06 and 6.07 and addition to aldehydes and ketones
Optimization of benzyl zinc additions
We began by investigating the addition of the unsubstituted benzyl zinc reagent 6.09 to the p-
methoxy- and p-methylcarboxy-substituted aromatic imine substrates 6.10 and 6.11, respectively
(Table 6.1). For benzyl zinc reagent 6.09, a 30 minute reagent preparation time was found to
produce the optimal results (entries 1 and 3). Extended reagent preparation time was found to
result in lower diastereoselectivity (entry 2) as well as overaddition into the ester functional
group (entry 4). Because appropriate reagent preparation time was found to vary with the quality
Table 6.1. Optimization of benzyl zinc reagent additions
entry preparation time imine product yield,a % dr
b
1 30 min 6.10 6.12 70 92:8
2 3 h 6.10 6.12 76 81:19
3 30 min 6.11 6.13 77 93:7
4 3 h 6.11 6.13 overadditionc nd
d
5e 30 min 6.11 6.13 83 90:10
6f 30 min 6.11 6.13 88 93:7
aDetermined by
1H NMR relative to 1,3,5-trimethoxybenzene as an external standard.
bDetermined by
1H
NMR. c57% triple addition product, 37% double addition product.
dNot determined.
eReagent stored in a
Schlenk flask under static N2 for 20 days. f4.0 equiv of 6.09.
126
of the magnesium used, reagent preparations were monitored by GC analysis, following
disappearance of benzyl chloride over time after quenching an aliquot of the reaction mixture
with water.
These negative effects were not observed when the reagent was filtered away from excess
Mg0 immediately after consumption of the benzyl chloride starting material (as determined by
GC analysis). Reagent stored in a Schlenk flask for 20 days reacted similarly to freshly prepared
reagent although a decrease in concentration from 0.33 M to 0.19 M10
was observed (entry 5).
Furthermore, overaddition was not observed with a large excess of benzyl zinc reagent prepared
under the standard conditions (entry 6). Together, these results suggest that the presence of
excess Mg0 after formation of the initial desired benzyl zinc reagent leads to the formation of a
second more reactive and less selective organometallic reagent.
Evaluation of substrate scope for diastereoselective benzyl zinc addition
We next evaluated a range of N-tert-butanesulfinyl aldimine substrates and organozinc
coupling partners under these optimal conditions (Table 6.2). Reactions with both electron-rich
(entry 1) and electron-poor aromatic imines (entries 2–5) proceeded with excellent yields and
good diastereoselectivity. Para, meta and ortho substitution were all well tolerated (entries 3–5).
As expected, ortho substitution (entry 5) resulted in a slightly diminished yield but proceeded
with very high diastereoselectivity. Additions to ester and nitrile substituted imines (entries 2, 7,
9, and 10) demonstrated the functional group compatibility of this method. Furthermore, the
benzyl reagent added to the 3-pyridyl imine substrate in high yield and with good
diastereoselectivity (entries 6 and 8), indicating that nitrogen heterocycles that often interfere
with transition metal catalyzed additions, such as Rh-catalyzed arylboronic acid additions,3 do
not interfere with these MgCl2-enhanced benzyl zinc reagent additions. Additions to alkyl imine
substrates (entries 11 and 12) also proceeded in good yields although with only moderate
selectivity. Finally, electron-neutral, electron-rich, and electron-poor organozinc coupling
partners reacted smoothly and provided good stereoselectivity and functional group tolerance
(entries 2, 7 and 9).
Further highlighting the functional group compatibility of this method, organozinc reagent
6.33, which contains an ester substituent, was added to both unsubstituted and nitrile-substituted
aromatic imines 6.34 and 6.20 with good yields and moderate to good selectivity (Scheme 6.3).
However, these reactions required the more reactive dibenzyl zinc reagent 6.07, prepared with
0.55 equiv of ZnCl2 (Scheme 6.2. Preparation of benzyl zinc reagents 6.06 and 6.07 and addition
to aldehydes and ketones).
Stereochemical rationale
The sense of induction for these addition reactions was determined by rigorously establishing
the absolute configurations of addition product 6.12 by chemical correlation and products 6.27
and 6.31 by X-ray structural analysis (Figure 6.1). The sense of induction is consistent with an
open transition state as proposed for a number of N-tert-butanesulfinyl imine addition reactions
(Scheme 6.4).1 Presumably, the excess of coordinating ions in conjunction with the use of a
coordinating solvent favor this transition state over a chelating transition state.
127
Table 6.2. Benzyl zinc additions to N-tert-butanesulfinyl aldimines
entry benzyl zinc X imine R product yield,a % dr
b
1 6.09 H 6.10 4-OMeC6H4 6.12 85 90:10c
2 6.09 H 6.11 4-CO2MeC6H4 6.13 86 92:8
3 6.09 H 6.16 4-ClMeC6H4 6.23 87 92:8
4 6.09 H 6.17 3-ClMeC6H4 6.24 86 92:8
5 6.09 H 6.18 2-ClMeC6H4 6.25 79 >99:1
6 6.09 H 6.19 3-Py 6.26 98 96:4
7 6.14 OMe 6.11 4-CO2MeC6H4 6.27 69 94:6d
8 6.14 OMe 6.19 3-Py 6.28 42 98:2
9 6.15 F 6.11 4-CO2MeC6H4 6.29 86 94:6
10 6.15 F 6.20 4-CNMeC6H4 6.30 83 98:2
11 6.15 F 6.21 tBu 6.31 77 76:24d,e
12 6.15 F 6.22 PhCH2CH2 6.32 76 77:23f
aIsolated yield of mixture of diastereomers after purification by chromatography.
bDetermined by HPLC
comparison to authentic diastereomers. cAbsolute configuration determined by comparison of the optical
rotation of the amine obtained upon sulfinyl deprotection to literature values (see experimental). dAbsolute configuration was determined by X-ray crystallography.
eDetermined by mass balance of
separately isolated diastereomers. fDetermined by
1H and
19F NMR.
Scheme 6.3. Ester-substituted dibenzyl zinc additions
Scheme 6.4. Stereochemical rationale for the reaction
128
Figure 6.1. Crystal structures of inhibitor 6.27 (a) and 6.31 (b).
Preparation of aspartyl protease inhibitor precursors
We also believed that this methodology offered an efficient new route to aspartyl protease
inhibitors including inhibitors of β-secretase (BACE 1) and HIV protease, which are under
development as potential treatments for Alzheimer’s disease and HIV, respectively.11
Two key
features made this class of molecules an attractive target. First, aspartyl protease inhibitors
commonly feature a hydroxyethylamine isostere of phenylalanine, which could be derived from
a glyceraldehyde-derived imine and a benzyl zinc reagent (Scheme 6.5). Second, the introduction
of functionalized benzyl rings has been utlilized to drive potency and to improve
pharmacokinetic properties.12
Scheme 6.5. Retrosynthetic approach to aspartyl protease inhibitors
To evaluate benzyl zinc additions for aspartyl protease inhibitor synthesis, we focused on the
synthesis of anti-3-amino-4-arylbutane-1,2-diol derivatives (Scheme 6.6). Benzyl zinc reagent
6.09 added to imine 6.37, prepared from isopropylidene-protected glyceraldehyde, in high yield
and with exceptionally high selectivity. Importantly, the stereochemistry obtained is that most
commonly found in hydroxyethylamine-based protease inhibitors. Addition to imine 6.40 also
proceeded in high yield but with modest selectivity for the syn diastereomer. For imine 6.40 the
face selectivity provided by the sulfinyl group presumably opposes and modestly overrides the
stereochemical bias provided by the -stereocenter, whereas for imine 6.37 both the sulfinyl
group and the -stereocenter favor the same product. Diastereomer 6.38 was readily converted to
N-Boc-3-amino-1,2-diol 6.39 by simultaneous deprotection of the sulfinyl and isopropylidene
protecting groups followed by Boc protection of the amine functionality. N-Boc-3-amino-1,2-
diols with the stereochemistry present in 6.39 provide direct access to hydroxyethylamine
inhibitors.13
Oxidative conversion of 6.39 to N-Boc-(S)-phenylalanine also enabled rigorous
assignment of the configuration at the amine stereocenter.
129
Scheme 6.6. Addition to glyceraldehyde-derived imines for the synthesis of 3-amino-1,2-diols
Conclusions
The MgCl2-enhanced addition of benzyl zinc reagents to N-tert-butanesulfinyl imines occurs
readily at ambient temperature. Good yields, selectivity, and functional group tolerance are
observed for a variety of aromatic imines. Moreover, ester-substituted dibenzyl zinc reagents add
to nitrile-substituted imines without cross-reactivity, further highlighting the functional-group
compatible nature of the transformation. Although benzyl additions to aliphatic imines generally
showed only moderate selectivity, addition to sulfinyl imine 6.37, prepared from isopropylidene-
protected glyceraldehyde, proceeded in high yield and with very high selectivity, indicating that
this method should allow the rapid introduction of a variety of functionalized benzyl substituents
into hydroxyethylamine-based aspartyl protease inhibitors.
Experimental
General synthetic methods
Unless otherwise noted, all reagents were obtained from commercial suppliers and used
without further purification. Benzyl chlorides were filtered through neutral Al2O3 (Brockman I)
immediately prior to use. Magnesium turnings were purchased new from Riedel-de Haën
(Sigma-Aldrich). Zinc chloride (H2O, oxide, OH < 100 ppm; 99.99% Zn) was purchased from
Strem, and lithium chloride, from Fluka (Sigma-Aldrich). Tetrahydrofuran (THF) was passed
through a column of activated alumina under nitrogen, and methanol was distilled from CaH2.
Salt solutions were prepared as previously described by Knochel and coworkers.7a
All reactions
involving air-sensitive and moisture-sensitive reagents were carried out using syringes, tripled
flushed with nitrogen before use. Syringe filtrations were performed with Millex-HN 0.45 µm
Nylon 33 mm syringe filters. Glassware was dried overnight at 140 ˚C or flame-dried under
vacuum prior to use. Chromatography was performed with Merck 60 230–240 mesh silica gel, or
utilizing a Biotage SP Flash Purification System (Biotage No. SP1-B1A). NMR spectra were
obtained at ambient temperature on a Bruker AVB-400 or AVB-500 spectrometer. NMR
130
chemical shifts are reported in ppm relative to CHCl3 (7.26), or TMS (0.00) for 1H,
trifluoroacetic acid (−76.55) for 19
F, and CDCl3 (77.16) or TMS (0.00) for 13
C. Determinations
of diastereomeric ratios were performed using an Agilent 1100 series HPLC equipped with a
normal-phase silica column (Microsorb Si 100 Å packing) and a multi-wavelength detector;
samples were dissolved in 3:1 hexanes:iPrOH. HPLC methods were developed using the authentic diastereomers of products 6.12–6.13, 6.23–6.30, 6.35–6.36 and 6.38 prepared from the
compound of interest14
or via unselective Grignard addition to imine 6.37. IR spectra were
recorded on a Nicolet 6700 FTIR spectrometer, and only partial data are provided. Melting
points were acquired using a Mel-Temp apparatus, and they are reported uncorrected. Specific
rotations were determined using a Perkin-Elmer 341 polarimeter with a sodium lamp, and
concentrations are reported in g/dL. Mass spectra (HRMS) analysis was performed by the Yale
Protein Expression Database facility on a 9.4T Bruker Qe FT-ICR MS.
Preparation of benzyl zinc reagents
General procedure. To an appropriately sized Schlenk flask was added magnesium turnings
(2.5 equiv). The flask was flushed with N2, and then a THF solution (1.0 mL/mmol of the benzyl
chloride) of ZnCl2 (1.1 M) and LiCl (1.5 M) was added via syringe. The appropriate benzyl
chloride (1.0 equiv) and THF (0.5 mL/mmol of the benzyl chloride) were combined in a separate
round bottom flask. The original Schlenk flask was placed in an ambient temperature water bath,
and the benzyl chloride solution was added dropwise via syringe. The water bath was removed
upon addition, and the heterogeneous reaction mixture was stirred vigorously for the specified
time. Reaction times were determined by GC analysis, using complete consumption of the
benzyl chloride as the endpoint. The reaction mixture was removed from the magnesium into a
syringe and then passed through a syringe filter into a second Schlenk flask for storage. Reagent
concentrations were determined via iodometric titration.10
Benzyl zinc reagent 6.09. The general procedure was followed with 669 mg (27.5 mmol) of
magnesium turnings, 11.0 mL of ZnCl2/LiCl solution, and 1.3 mL (11 mmol) of benzyl chloride
in 5.5 mL of THF. The heterogeneous reaction mixture was stirred 30 min after addition of the
benzyl chloride solution. After transfer, iodometric titration determined a concentration of 0.43
M.
4-Methoxybenzyl zinc reagent 6.14. The general procedure was followed with 243 mg
(10.0 mmol) of magnesium turnings, 4.0 mL of ZnCl2/LiCl solution, and 0.54 mL (4.0 mmol) of
4-methoxybenzyl chloride in 2.0 mL of THF. The heterogeneous reaction mixture was stirred for
30 min after addition of the benzyl chloride solution. After transfer, iodometric titration
determined a concentration of 0.39 M.
131
4-Fluorobenzyl zinc reagent 6.15. The general procedure was followed with 608 mg (25.0
mmol) of magnesium turnings, 10.0 mL of ZnCl2/LiCl solution, and 1.20 mL (10.0 mmol) of 4-
fluorobenzyl chloride in 5.0 mL of THF. The heterogeneous reaction mixture was stirred for 35
min after addition of the benzyl chloride solution. After transfer, iodometric titration determined
a concentration of 0.36 M.
Di(3-ethylcarboxybenzyl) zinc reagent 6.33. To a 100 mL Schlenk flask was added 1.65 g
(68.0 mmol, 2.50 equiv) of magnesium turnings. The flask was flushed with N2, and then a THF
solution (13.6 mL) of ZnCl2 (1.1 M, 0.55 equiv) and LiCl (1.5 M, 0.75 equiv) was added. Ethyl
3-(chloromethyl)benzoate (4.60 mL, 27.2 mmol, 1.0 equiv) and THF (24.0 mL) were combined
in a separate round bottom flask. The original Schlenk flask was placed in an ambient
temperature water bath. The benzyl chloride solution was added dropwise via cannula over 8
min. Due to a strong exotherm, the reaction flask remained in the water bath while the reaction
mixture was stirred vigorously for 35 min, the endpoint determined from GC analysis. The
reaction mixture was removed from the magnesium via cannula filtration into a second Schlenk
flask. A 1.0 mL aliquot of reagent was stirred with 1.0 mL of 1.1 M ZnCl2 / 1.5 M LiCl solution
of THF, at which point, iodometric titration determined a concentration of 0.22 M of the
monobenzyl zinc species, and thus 0.11 M of the dibenzyl reagent.
Synthesis of N-tert-butanesulfinyl imine starting materials
Sulfinyl imines 6.10, 6.16, 6.19, 6.34,15
6.2116
and 6.2017
were synthesized according to
literature procedures.
General Procedure. Cs2CO3 (1.2 equiv) and tert-butanesulfinamide (1.2 equiv) were added
to an appropriately sized round bottom flask. The flask was flushed with N2 followed by the
addition of CH2Cl2 and the aldehyde (1.0 equiv, 0.2 M). The flask was equipped with a reflux
condenser, and the reaction mixture was refluxed in a 46 °C oil bath for 14–18 h. The mixture
was filtered through a pad of Celite and concentrated under reduced pressure. The desired
product was isolated via silica gel chromatography, visualizing by UV.
Sulfinyl imine 6.11. The general procedure was followed with 2.38 g (7.31 mmol) of
Cs2CO3, 0.886 g (7.31 mmol) of R-(–)-tert-butanesulfinamide and 1.00 g (6.09 mmol) of 4-
carbomethoxybenzaldehyde to afford the imine product 6.11 (1.53 g, 94%) as a white solid, m.p.
132
92.7–93.2 °C. IR (in CHCl3): 2955, 1724, 1280, 1109, 1084 cm–1
. []20
D –71.1 (c 0.78, CHCl3).
1H NMR (400 MHz, CDCl3): 8.63 (s, 1H), 8.13 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 8.4 Hz, 2H),
3.94 (s, 3H), 1.27 (s, 9H); 13
C NMR (101 MHz, CDCl3): 166.47, 162.07, 137.70, 133.43,
130.32, 129.38, 58.33, 52.65, 22.85. HRMS (m/z): [M+H]+ calcd. for C13H18NO3S, 268.1002;
found, 268.0999.
Sulfinyl imine 6.17. The general procedure was followed with 3.90 g (12.0 mmol) of
Cs2CO3, 1.45 g (12.0 mmol) of R-(–)-tert-butanesulfinamide and 1.13 mL (10.0 mmol) of 3-
chlorobenzaldehyde to afford the imine product 6.17 (2.40 g, 98%) as an off-white solid, m.p.
34.5–35.3 °C. IR (in CHCl3): 2981, 1601, 1566, 1212, 1082 cm–1
. []20
D –77.3 (c 1.27, CHCl3).
1H NMR (400 MHz, CDCl3): 8.48 (s, 1H), 7.80 (s, 1H), 7.63 (d, J = 7.5 Hz, 1H), 7.41 (d, J =
8.7 Hz, 1H), 7.35 (apparent t, J = 7.8 Hz, 1H), 1.21 (s, 9H); 13
C NMR (101 MHz, CDCl3): δ
161.48, 135.68, 135.18, 132.34, 130.31, 128.63, 127.96, 58.02, 22.66. HRMS (m/z): [M+H]+
calcd. for C11H15NOS[35]
Cl, 244.0557; found, 244.0553.
Sulfinyl imine 6.18. The general procedure was followed with 3.90 g (12.0 mmol) of
Cs2CO3, 1.45 g (12.0 mmol) of R-(–)-tert-butanesulfinamide and 1.13 mL (10.0 mmol) of 2-
chlorobenzaldehyde to afford the imine product 6.18 (2.36 g, 97%) as a white solid. Analytical
data were consistent with previous literature reports.18
Sulfinyl imine 6.22. To a 100 mL round bottom flask was added 1.92 g (12.0 mmol) CuSO4
and 1.45 g (12.0 mmol) of R-(–)-tert-butanesulfinamide. The flask was flushed with N2, then
CH2Cl2 (50 mL) was added, followed by 3-phenylpropanal (90% purity, 1.51 mL, 10.0 mmol).
The suspension was stirred vigorously for 22h then filtered through Celite and concentrated
under vacuum. Purification by flash chromatography afforded imine product 6.22 (0.961 g, 40%)
as a clear oil. Analytical data were consistent with previous literature reports.19
133
Addition of benzyl zinc reagents to N-tert-butanesulfinyl imines
General Procedure. A 25-mL round bottom flask was charged with imine (0.500 mmol) and
flushed with N2. The imine was then dissolved in THF (0.50 mL). The benzyl zinc reagent was
added dropwise via syringe, and the reaction mixture was stirred 24 h at ambient temperature.
The reaction mixture was then cooled to 0 °C, diluted with EtOAc, and the reaction was
quenched with 15 mL of sat. NH4Cl (aq). The aqueous layer was extracted with EtOAc (3 × 20
mL), and the combined organic layers were dried over NaSO4, filtered, and concentrated under
reduced pressure. The desired product was isolated using silica gel chromatography, visualizing
by UV and PMA staining. All products were isolated as a mixture of diastereomers, and NMR
data corresponds to the major diastereomer unless otherwise noted.
Product 6.12. The general procedure was followed with 120 mg (0.500 mmol) of imine 6.10
and 2.8 mL (1.0 mmol) of freshly prepared benzyl zinc reagent 6.09 (0.36 M). Purification by
flash chromatography afforded product 6.12 (142 mg, 85%, 90:10 dr) as a white solid, m.p. 92.9–94.4 °C. HPLC (silica column, hexanes:EtOH, 99:1 to 98:2 over 55 min, 0.5 mL/min, λ =
210 nm): tr,minor = 14.7 min, tr,major = 11.7 min. IR (in CHCl3): 3330, 2960, 1425, 1051, 1023
cm–1
. 1H NMR (400 MHz, CDCl3): δ 7.23–7.12 (m, 5H), 7.03–6.93 (m, 2H), 6.82 (d, J = 8.7 Hz,
2H), 4.70–4.35 (m, 1H), 3.78 (s, 3H), 3.46 (d, J = 3.6 Hz, 1H), 3.28 (dd, J = 13.4, 6.5 Hz, 1H), 2.98 (dd, J = 13.4, 7.8 Hz, 1H), 1.15 (s, 9H);
13C NMR (126 MHz, CDCl3): δ 159.27, 137.76,
133.90, 129.81, 128.63, 128.33, 126.52, 114.00, 60.37, 55.99, 55.37, 43.70, 22.68. HRMS (m/z):
[M+H]+ calcd. for C19H26NO2S, 332.1679; found, 332.1675.
Free amine 6.42. (Note: compound 6.42 was synthesized to determine sense of induction of
the benzyl zinc addition through chemical correlation). A flame-dried scintillation vial was
charged sulfinyl amine 6.12 (20.7 mg, 0.0624 mmol, 93:7 dr). The product was dissolved in dry
MeOH (4.0 mL), and 4.0 M HCl in dioxane (80 L, 0.32 mmol) was added dropwise via syringe.
The reaction mixture was stirred 3 h at ambient temperature. The reaction mixture was then
concentrated under reduced pressure, diluted with EtOAc, and washed with sat. NaHCO3 (aq).
The aqueous layer was extracted twice more with EtOAc. The combined organic layers were
dried over MgSO4, filtered, and concentrated under reduced pressure. The product was isolated
as a clear, yellow oil (11.9 mg, 87%) and used without further purification. Analytical data were
consistent with those previously reported. []20
D –97.0 (c 0.70, 1:1 MeOH:0.1N HCl) [lit. []20
D
–91.9 (c 1.00, 85% ee, 1:1 MeOH:0.1N HCl)].20
134
Product 6.13. The general procedure was followed with 134 mg (0.500 mmol) of imine 6.11
and 2.3 mL (1.0 mmol) of freshly prepared benzyl zinc reagent 6.09 (0.43 M). Purification by
flash chromatography afforded product 6.13 (154 mg, 86%, 92:8 dr) as a clear, amorphous solid. HPLC (silica column, 98.5:1.5 hexanes:EtOH, 1.0 mL/min, λ = 254 nm): tr,minor = 20.8 min,
tr,major = 23.3 min. IR (in CHCl3): 3240, 2953, 1718, 1275, 1046 cm–1
. 1H NMR (400 MHz,
CDCl3): δ 7.97 (d, J = 8.3 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.24–7.14 (m, 3H), 7.00 (dd, J =
7.6, 1.4 Hz, 2H), 4.78–4.53 (m, 1H), 3.90 (s, 3H), 3.75 (d, J = 4.8 Hz, 1H), 3.30 (dd, J = 13.4, 6.7 Hz, 1H), 3.02 (dd, J = 13.4, 7.5 Hz, 1H), 1.16 (s, 9H);
13C NMR (101 MHz, CDCl3): δ
166.67, 146.89, 136.83, 129.74, 129.51, 129.47, 128.25, 127.26, 126.59, 60.61, 56.09, 52.03,
43.53, 22.41. HRMS (m/z): [M+Na]+ calcd. for C20H25NO3SNa, 382.1447; found, 382.1429.
Product 6.23. The general procedure was followed with 122 mg (0.500 mmol) of imine 6.16
and 3.1 mL (1.0 mmol) of freshly prepared benzyl zinc reagent 6.09 (0.32 M). Purification by
flash chromatography afforded product 6.23 (146 mg, 87%, 92:8 dr) as a white solid, m.p. 129.7–130.6 °C. HPLC (silica column, 95:5 hexanes:iPrOH, 0.5 mL/min, λ = 210 nm): tr,minor =
14.7 min, tr,major = 11.7 min. IR (in CHCl3): 3201, 2957, 1493, 1043, 1015 cm–1
. 1H NMR (500
MHz, CDCl3): δ 7.26 (t, J = 4.2 Hz, 3H), 7.18 (m, 4H), 7.05–6.90 (m, 2H), 4.57 (m, 1H), 3.50 (d,
J = 3.9 Hz, 1H), 3.27 (dd, J = 13.4, 6.5 Hz, 1H), 2.97 (dd, J = 13.4, 7.7 Hz, 1H), 1.15 (s, 9H); 13
C
NMR (126 MHz, CDCl3): δ 140.22, 136.99, 133.59, 129.60, 128.70, 128.33, 126.64, 60.24,
56.05, 43.55, 22.49. HRMS (m/z): [M+H]+ calcd. for C18H23NOS
[35]Cl, 336.1183; found,
336.1184.
Product 6.24. The general procedure was followed with 122 mg (0.500 mmol) of imine 6.17
and 2.3 mL (1.0 mmol) of freshly prepared benzyl zinc reagent 6.09 (0.43 M). Purification by
flash chromatography afforded product 6.24 (144 mg, 86%, 92:8 dr) as a white solid, m.p.
116.7–117.4 °C. HPLC (silica column, 95:5 hexanes:iPrOH, 0.5 mL/min, λ = 210 nm): tr,minor =
14.3 min, tr,major = 12.4 min. IR (in CHCl3): 3172, 2963, 1597, 1041, 1016 cm–1
. 1H NMR (400
MHz, CDCl3): δ 7.28–7.15 (m, 6H), 7.12–7.06 (m, 1H), 7.04–6.96 (m, 2H), 4.62–4.50 (m, 1H),
135
3.52 (br s, 1H), 3.26 (dd, J = 13.5, 6.7 Hz, 1H), 2.99 (dd, J = 13.4, 7.5 Hz, 1H), 1.15 (s, 9H); 13
C NMR (126 MHz, CDCl3): δ 143.95, 137.06, 134.55, 129.94, 129.74, 128.49, 128.21, 127.44,
126.83, 125.85, 60.57, 56.26, 43.68, 22.63. HRMS (m/z): [M+H]+ calcd. for C18H23NOS
[35]Cl,
336.1183; found, 336.1187.
Product 6.25. The general procedure was followed with 122 mg (0.500 mmol) of imine 6.18
and 2.3 mL (1.0 mmol) of freshly prepared benzyl zinc reagent 6.09 (0.43 M). Purification by
flash chromatography afforded product 6.25 (133 mg, 79%, >99:1 dr) as a white solid, m.p. 109.4–110.2 °C. HPLC (silica column, 95:5 hexanes:iPrOH, 0.5 mL/min, λ = 210 nm): tr,minor =
13.9 min, tr,major = 15.2 min. IR (in CHCl3): 3192, 2958, 2922, 1454, 1029 cm–1
. 1H NMR (500
MHz, CDCl3): δ 7.44–7.31 (m, 2H), 7.31–7.18 (m, 5H), 7.12 (d, J = 7.0 Hz, 2H), 5.15–4.92 (m,
1H), 3.76 (d, J = 6.3 Hz, 1H), 3.19 (dd, J = 13.7, 5.5 Hz, 1H), 3.05 (dd, J = 13.7, 8.2 Hz, 1H), 1.06 (s, 9H).
13C NMR (126 MHz, CDCl3): δ 139.25, 137.26, 132.82, 129.83, 129.65, 128.80,
128.66, 128.39, 127.05, 126.76, 58.29, 56.37, 42.65, 22.33. HRMS (m/z): [M+Na]+ calcd. for
C18H22NOS[35]
ClNa, 358.1003; found, 358.1000.
Product 6.26. The general procedure was followed with 105 mg (0.500 mmol) of imine 6.19
and 2.8 mL (1.0 mmol) of freshly prepared benzyl zinc reagent 6.09 (0.36 M). Purification by
flash chromatography afforded product 6.26 (149 mg, 98%, 96:4 dr), which became a waxy solid
upon storage at 0 °C. HPLC (silica column, 85:15 hexanes (0.1% v/v Et2NH):iPrOH, 0.5
mL/min, λ = 254 nm): tr,minor = 18.8 min, tr,major = 25.6 min. IR (in CHCl3): 3204, 2957, 1476,
1428, 1052 cm–1
. 1H NMR (400 MHz, CDCl3): δ 8.60–8.40 (m, 2H), 7.59–7.46 (m, 1H), 7.25–
7.13 (m, 4H), 7.07–6.89 (m, 2H), 4.72–4.52 (m, 1H), 3.63 (d, J = 4.3 Hz, 1H), 3.32 (dd, J = 13.4, 6.7 Hz, 1H), 3.00 (dd, J = 13.4, 7.7 Hz, 1H), 1.14 (s, 9H);
13C NMR (126 MHz, CDCl3): δ
149.35, 149.04, 137.23, 136.69, 135.12, 129.69, 128.54, 126.91, 123.45, 58.82, 56.28, 43.48,
22.57. HRMS (m/z): [M+H]+ calcd. for C17H23N2OS, 303.1526; found, 303.1526.
136
Product 6.27. The general procedure was followed with 120 mg (0.50 mmol) of imine 6.11
and 2.6 mL (1.0 mmol) of freshly prepared benzyl zinc reagent 6.14 (0.39 M). Purification by
flash chromatography afforded product 6.27 (134 mg, 69%, 96:4 dr) as a white solid, m.p. 108.6–111.8 °C. HPLC (silica column, 98.5:1.5 hexanes:EtOH, 0.5 mL/min, λ = 210 nm): tr,minor
= 39.2 min, tr,major = 42.0 min. IR (neat): 2960, 1716, 1514, 1276, 1246, 1055 cm–1
. 1H NMR
(400 MHz, CDCl3): δ 7.88 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.2 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H),
6.65 (d, J = 8.4 Hz, 2H), 4.60–4.50 (m, 1H), 3.83 (s, 3H), 3.68 (s, 3H), 3.52 (d, J = 3.9 Hz, 1H),
3.18 (dd, J = 13.5, 6.2 Hz, 1H), 2.87 (dd, J = 13.5, 7.8 Hz, 1H), 1.10 (s, 9H); 13
C NMR (101 MHz, CDCl3): δ 166.98, 158.49, 147.07, 130.76, 129.97, 129.75, 128.85, 127.58, 113.87, 60.68,
56.30, 55.36, 52.31, 42.80, 22.70. HRMS (m/z): [M+H]+ calcd. for C21H28NO4S, 390.1734;
found, 390.1732.
Product 6.28. The general procedure was followed with 105 mg (0.500 mmol) of imine 6.19
and 3.3 mL (1.0 mmol) of freshly prepared benzyl zinc reagent 6.14 (0.30 M). Purification by
flash chromatography afforded product 6.28 (69.6 mg, 42%, 98:2 dr) which became a waxy,
yellow solid upon storage at 0 °C. HPLC (silica column, 85:15 hexanes (0.1% v/v EtN2H):
iPrOH, 0.5 mL/min, λ = 210 nm): tr,minor = 13.9 min, tr,major = 15.2 min. IR (in CHCl3): 2961,
2925, 1513, 1249, 1040 cm–1
. 1H NMR (400 MHz, CDCl3): δ 8.54–8.42 (m, 2H), 7.63–7.39 (m,
1H), 7.26–7.14 (m, 1H), 6.92–6.75 (m, 2H), 6.75–6.64 (m, 2H), 4.62–4.42 (m, 1H), 3.69 (s, 3H),
3.66 (d, J = 4.9 Hz, 1H), 3.19 (dd, J = 13.6, 6.6 Hz, 1H), 2.89 (dd, J = 13.6, 7.6 Hz, 1H), 1.09 (s, 9H);
13C NMR (126 MHz, CDCl3): δ 158.62, 148.98, 148.83, 138.11, 136.18, 130.72, 128.44,
123.88, 114.03, 58.94, 56.40, 55.34, 42.63, 22.63. HRMS (m/z): [M+H]+ calcd. for
C18H25N2O2S, 333.1631; found, 333.1621.
Product 6.29. The general procedure was followed with 120 mg (0.50 mmol) of imine 6.11
and 3.3 mL (1.0 mmol) of freshly prepared benzyl zinc reagent 6.15 (0.30 M). Purification by
flash chromatography afforded product 6.29 (163 mg, 86%, 94:6 dr) as an amorphous solid.
137
HPLC (silica column, 99:1 hexanes:EtOH, 1.0 mL/min, λ = 210 nm): tr,minor = 24.1 min, tr,major =
27.8 min. IR (neat): 3238, 2954, 1718, 1509, 1276, 1046 cm–1
. 1H NMR (400 MHz, CDCl3): δ
7.95 (d, J = 8.3 Hz, 2H), 7.26 (d, J = 8.3 Hz, 2H), 6.93–6.83 (m, 4H), 4.65–4.58 (m, 1H), 3.90 (s,
3H), 3.55 (d, J = 4.1 Hz, 1H), 3.30 (dd, J = 13.5, 6.2 Hz, 1H), 2.96 (dd, J = 13.5, 7.9 Hz, 1H), 1.18 (s, 9H);
13C NMR (101 MHz, CDCl3): δ 166.90, 146.66, 132.65 (d, JCF = 3.1 Hz), 131.24
(d, JCF = 7.8 Hz), 130.08, 129.97, 127.54, 115.44, 115.23, 60.56, 56.32, 52.37, 42.72, 22.71; 19
F NMR (376 MHz, CDCl3) δ –116.44. HRMS (m/z): [M+Na]
+ calcd. for C20H24NO3FSNa,
400.1353; found, 400.1345.
Product 6.30. The general procedure was followed with 117 mg (0.50 mmol) of imine 6.20
and 2.7 mL (1.0 mmol) of freshly prepared benzyl zinc reagent 6.15 (0.37 M). Purification by
flash chromatography afforded product 6.30 (143 mg, 83%, 97:3 dr) as a white solid, m.p. 118.2–119.5 °C. HPLC (silica column, 90:10 hexanes:iPrOH, 0.5 mL/min, λ = 210 nm): tr,minor =
14.2 min, tr,major = 12.8 min. IR (neat): 3236, 2954, 2225, 1509, 1044 cm–1
. 1H NMR (400 MHz,
CDCl3): δ 7.60 (d, J = 8.1 Hz, 2H), 7.32 (d, J = 8.2 Hz, 2H), 6.93–6.89 (m, 4H), 4.65–4.58 (m,
1H), 3.53 (d, J = 4.6 Hz, 1H), 3.28 (dd, J = 13.7, 6.4 Hz, 1H), 2.96 (dd, J = 13.6, 7.7 Hz, 1H), 1.17 (s, 9H);
13C NMR (126 MHz, CDCl3): δ 146.98, 132.63, 132.26 (d, JCF = 3.2 Hz), 131.21
(d, JCF = 8.1 Hz), 128.30, 118.71, 115.65, 115.49, 112.15, 60.72, 56.52, 42.74, 22.69; 19
F NMR (376 MHz, CDCl3): δ –115.92. HRMS (m/z): [M+H]
+ calcd. for C19H22N2OFS, 345.1431; found,
345.1429.
Product 6.31. The general procedure was followed with 95 mg (0.50 mmol) of imine 6.21
and 2.8 mL (1.0 mmol) of freshly prepared benzyl zinc reagent 6.15 (0.36 M). Purification by
flash chromatography afforded separable diastereomeric products 6.31a (major) and 6.31b
(minor) in a 76:24 dr. 6.31a (RS,R): 88 mg, 59%, white solid, m.p. 117.4–118.3 °C. IR (neat):
2956, 1510, 1459, 1214, 1043 cm–1
. 1H NMR (400 MHz, CDCl3): δ 7.14–7.09 (m, 2H), 6.98–
6.91 (m, 2H), 3.26 (m, 1H), 3.11 (d, J = 7.2 Hz, 1H), 3.03 (dd, J = 14.2, 2.4 Hz, 1H), 2.43 (dd, J = 14.2, 10.6 Hz, 1H), 1.06 (s, 9H), 0.92 (s, 9H);
13C NMR (101 MHz, CDCl3): δ 135.67 (d, JCF =
3.2 Hz), 131.12 (d, JCF = 7.9 Hz), 115.34, 115.13, 67.38, 56.25, 37.95, 35.18, 27.35, 22.63; 19
F NMR (376 MHz, CDCl3): δ –117.63. HRMS (m/z): [M+H]
+ calcd. for C16H27NOFS, 300.1792;
found, 300.1788. 6.31b (RS,S): 28 mg, 18%, white solid, m.p. 90.8–91.5 °C. IR (in CHCl3):
2960, 1510, 1476, 1223, 1062 cm–1
. 1H NMR (400 MHz, CDCl3): δ 7.25–7.19 (m, 2H), 7.02–
6.96 (m, 2H), 3.24 (m, 1H), 3.15–3.02 (m, 2H), 2.63 (dd, J = 14.6, 8.4 Hz, 1H), 1.14 (s, 9H),
138
0.96 (s, 9H); 13
C NMR (101 MHz, CDCl3): δ 134.50 (d, JCF = 3.3 Hz), 131.22 (d, JCF = 7.7 Hz),
115.55, 115.34, 66.04, 56.55, 37.58, 36.21, 27.12, 22.96; 19
F NMR (376 MHz, CDCl3): δ
–116.75. HRMS (m/z): [M+H]+ calcd. for C16H27NOFS, 300.1792; found, 300.1787.
Product 6.32. The general procedure was followed with 118 mg (0.50 mmol) of imine 6.22
and 2.8 mL (1.0 mmol) of freshly prepared benzyl zinc reagent 6.15 (0.36 M). Purification by
flash chromatography afforded product 6.32 (131 mg, 76%) as a clear oil. IR (in CHCl3): 3224, 2926, 1510, 1221, 1052 cm
–1.
1H NMR (400 MHz, CDCl3): δ 7.27–7.16 (m, 2H), 7.16–7.09 (m,
3H), 7.09–7.02 (m, 2H), 6.96–6.85 (m, 2H), 3.43–3.33 (m, 1H), 3.05 (d, J = 6.2 Hz, 1H), 2.79–2.63 (m, 4H), 1.97–1.80 (m, 2H), 1.03 (s, 9H);
13C NMR (101 MHz, CDCl3): δ 141.45, 134.18
(d, JCF = 3.1 Hz), 131.22 (d, JCF = 7.8 Hz), 128.68, 128.67, 126.22, 115.45, 115.24, 57.98, 56.03,
42.02, 37.10, 32.22, 22.72; 19
F NMR (376 MHz, CDCl3): –117.04. HRMS (m/z): [M+H]+ calcd.
for C20H27NOFS, 348.1792; found, 348.1774.
Product 6.35. The general procedure was followed with 110 mg (0.53 mmol) of imine 6.34
and 4.8 mL (1.1 mmol) of freshly prepared dibenzyl reagent 6.33 (0.22 M). Purification by flash
chromatography afforded product 6.35 (145 mg, 73%, 80:20 dr) as an oil. HPLC (silica column, 93:7 hexanes:iPrOH, 0.5 mL/min, λ = 210 nm): tr,minor = 16.7 min, tr,major = 14.8 min. IR (in
CHCl3): 3018, 2983, 1713, 1281, 1214 cm–1
. 1H NMR (400 MHz, CDCl3): δ 7.78 (d, J = 7.8 Hz,
1H), 7.65 (s, 1H), 7.28–7.10 (m, 6H), 7.03 (d, J = 7.6 Hz, 1H), 4.59–4.51 (m, 1H), 4.28 (q, J =
7.1 Hz, 2H), 3.48 (d, J = 3.3 Hz, 1H), 3.27 (dd, J = 13.5, 6.4 Hz, 1H), 3.00 (dd, J = 13.4, 7.7 Hz, 1H), 1.31 (t, J = 7.1 Hz, 3H), 1.10 (s, 9H);
13C NMR (101 MHz, CDCl3): δ 166.70, 141.49,
137.89, 134.42, 130.91, 130.53, 128.82, 128.34, 128.22, 127.93, 127.45, 61.13, 60.69, 56.18,
43.36, 22.75, 14.53. HRMS (m/z): [M+H]+ calcd. for C21H28NO3S, 374.1784; found, 374.1776.
Product 6.36. The general procedure was followed with 117 mg (0.50 mmol) of imine 6.20
and 4.6 mL (1.0 mmol) of freshly prepared dibenzyl reagent 6.33 (0.22 M). Purification by flash
139
chromatography afforded product 6.36 (147 mg, 74%, 97:3) as an amorphous solid. HPLC (silica column, 88:12 hexanes:iPrOH, 0.5 mL/min, λ = 210 nm): tr,minor = 17.2 min, tr,major = 20.8 min.
IR (in CHCl3): 3246, 3018, 2982, 1715, 1281, 1214, 1055 cm–1
. 1H NMR (400 MHz, CDCl3): δ
7.87 (d, J = 7.8 Hz, 1H), 7.70 (s, 1H), 7.58 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.3 Hz, 2H), 7.28 (t,
J = 7.6 Hz, 1H), 7.10 (d, J = 7.7 Hz, 1H), 4.70–4.64 (m, 1H), 4.35 (q, J = 7.1 Hz, 2H), 3.59 (d, J
= 5.2 Hz, 1H), 3.32 (dd, J = 13.6, 6.8 Hz, 1H), 3.06 (dd, J = 13.6, 7.4 Hz, 1H), 1.38 (t, J = 7.1 Hz, 3H), 1.15 (s, 9H);
13C NMR (101 MHz, CDCl3): δ 166.47, 146.89, 136.90, 134.20, 132.63,
130.83, 130.72, 128.66, 128.34, 128.23, 118.70, 112.08, 61.29, 60.67, 56.55, 43.22, 22.65, 14.52.
HRMS (m/z): [M+H]+ calcd. for C22H27N2O3S, 399.1737; found, 399.1731.
Synthesis of aspartyl protease inhibitor precursors
Sulfinyl imine 6.37. To a 250 mL round bottom flask was added 505 mg (3.88 mmol) of
(2R)-2,3-O-isopropylideneglyceraldehyde21
and 699 mg (5.77 mmol) of S-(+)-tert-
butanesulfinamide. The flask was flushed very briefly with N2, then CH2Cl2 (96 mL) and TiOEt4
(4.0 mL, 19.2 mmol) were added. The reaction mixture was stirred for 6 h. The flask was then
cooled to 0 °C, and 4 mL of cold water was added. The resulting mixture was filtered through
Celite, washing with EtOAc. The filtrate was washed with 20 mL of brine, and the aqueous layer
was extracted twice more with EtOAc. The combined organic layers were then concentrated
under vacuum. Purification by flash chromatography afforded imine product 6.37 (584 mg, 65%)
as a light yellow oil. IR (neat): 2984, 1625, 1372, 1219, 1061 cm–1
. 1H NMR (400 MHz, CDCl3):
δ 8.05 (d, J = 4.1 Hz, 1H), 4.98–4.66 (m, 1H), 4.42–4.12 (m, 1H), 4.03 (dd, J = 8.6, 5.1 Hz, 1H),
1.44 (s, 3H), 1.41 (s, 3H), 1.19 (s, 9H); 13
C NMR (101 MHz, CDCl3): δ 168.15, 110.97, 77.07,
67.37, 57.16, 26.54, 25.57, 22.44. HRMS (m/z): [M+H]+ calcd. for C10H20NO3S, 234.11584;
found, 234.11580.
Product 6.38. The general procedure for additions of benzyl zinc reagents to N-tert-
butanesulfinyl imines was followed with 120 mg (0.516 mmol) of imine 6.37 and 3.1 mL (1.0
mmol) of freshly prepared benzyl zinc reagent 6.09 (0.33 M). Purification by flash
chromatography afforded product 6.38 (146 mg, 87%, >99:1 dr) as an off-white solid, m.p. 113.1–114.4 °C. HPLC (silica column, hexanes:iPrOH, 95:5, 0.5 mL/min, λ = 254 nm): tr,minor =
27.0 min (only observed in authentic diastereomer mixture prepared by Grignard reagent
addition), tr,major = 24.7 min. IR (in CHCl3) 3221, 1455, 1370, 1062 cm–1
. 1H NMR (400 MHz,
CDCl3): δ 7.42–7.07 (m, 5H), 4.20–4.12 (m, 1H), 4.12–4.04 (m, 1H), 3.96 (dd, J = 8.4, 6.1 Hz,
1H), 3.74–3.61 (m, 1H), 3.48 (d, J = 6.1 Hz, 1H), 3.01 (dd, J = 14.0, 7.4 Hz, 1H), 2.77 (dd, J = 14.0, 6.2 Hz, 1H), 1.48 (s, 3H), 1.33 (s, 3H), 1.11 (s, 9H);
13C NMR (101 MHz, CDCl3): δ
140
137.50, 129.67, 128.64, 126.73, 109.50, 65.99, 58.64, 56.23, 37.76, 26.64, 24.95, 22.61. HRMS
(m/z): [M+H]+ calcd. for C17H28NO3S, 326.1784; found, 326.1774.
Compound 6.39. A 25 mL round bottom flask was charged with product 6.38 (97 mg, 0.30
mmol). The flask was placed in an ambient temperature water bath, and a 4.0 M solution of HCl
in dioxane (6.0 mL, 24 mmol) was added. The bath was removed, and the reaction mixture was
stirred. After 10 min, 21.6 L of water (1.20 mmol) was added, and the reaction mixture was
stirred for 20 h. Et2O (10 mL) was then added slowly to the solution. The resulting white
precipitate was collected by vacuum filtration and washed with Et2O. The precipitation and
collection steps were repeated twice more on the filtrate with 5 mL of Et2O, concentrating the
sample at atmospheric pressure before the final precipitation. The precipitate was dissolved in
MeOH, transferred to a 25 mL round bottom flask, and concentration under vacuum to afford the
amine hydrochloride product (62.8 mg). Dioxane (3.75 mL), water (0.188 mL, 5% v/v) and
Boc2O (73.1 L, 0.328 mmol) were added to the flask. The flask was placed in a water bath, and
NEt3 (48.4 L, 0.347 mmol) was added. The flask was removed from the water bath, and the
reaction mixture was stirred 3 h at ambient temperature. The reaction mixture was then
concentrated under vacuum. Purification by flash chromatography afforded product 6.39 (74 mg,
86%) as a white solid. Analytical data were consistent with previous literature reports.22
1H NMR
(400 MHz, CDCl3): δ 7.45–7.11 (m, 5H), 4.55 (d, J = 8.6 Hz, 1H), 3.87–3.76 (m, 1H), 3.65 (dd,
J = 10.7, 7.9 Hz, 2H), 3.50–3.27 (m, 2H), 3.10 (dd, J = 14.2, 4.1 Hz, 1H), 2.91 (dd, J = 14.2, 7.7 Hz, 1H), 2.77 (d, J = 9.0 Hz, 1H), 1.38 (s, 9H);
13C NMR (101 MHz, CDCl3): δ 157.21, 137.43,
129.59, 128.78, 126.79, 80.57, 73.19, 63.02, 52.38, 36.60, 28.38.
N-(tert-Butoxycarbonyl)-L-phenylalanine (6.43). (Note: compound 6.43 was synthesized to
determine sense of induction of the benzyl zinc addition through chemical correlation). To a 10
mL round bottom flask was added 25 mg (0.089 mmol) of 6.39. Dioxane (0.40 mL) and water
(0.160 mL) were added, followed by Na2CO3 (5.8 mg, 0.055 mmol), KMnO4 (3.0 mg, 0.019
mmol), and NaIO4 (79 mg, 0.37 mmol) were added. The reaction mixture was stirred for 17 h.
The reaction mixture was then diluted with 5 mL of EtOAc, and the resulting solution was
washed with sat. NaHCO3 (aq.). The aqueous layer was washed with EtOAc and then acidified
with NaHSO4 (aq.) to pH 1, and was extracted with EtOAc (3 × 20 mL). The combined organic
layers were then concentrated under vacuum. Due to the presence of impurities, a second acid-
base extraction was required to afford amino acid product 6.43 (10 mg, 42%) as a clear oil.
Analytical data were consistent with previous literature reports.23
[]20
D +21.3 (c 0.667, EtOH)
[lit. []20
D +23.85 (c 2, EtOH)
24 and []20
D +25.2 (c 1, EtOH)
25].
141
Sulfinyl imine 6.40. Imine 6.40 was prepared analogously to imine 6.38 utilizing R-(–)-tert-
butanesulfinamide. The reaction afforded imine product 6.40 (439 mg, 48%) as a light yellow oil. IR (neat): 2984, 1626, 1372, 1218, 1059 cm
–1.
1H NMR (400 MHz, CDCl3): δ 8.02 (d, J =
4.6 Hz, 1H), 4.94–4.74 (m, 1H), 4.43–4.17 (m, 1H), 4.01 (dd, J = 8.7, 5.5 Hz, 1H), 1.46 (s, 3H), 1.41 (s, 3H), 1.20 (s, 9H);
13C NMR (101 MHz, CDCl3): δ 167.40, 111.04, 76.85, 67.18, 57.22,
26.43, 25.46, 22.40. HRMS (m/z): [M+H]+ calcd. for C10H20NO3S, 234.11584; found,
234.11580.
Product 6.41. The general procedure for additions of benzyl zinc reagents to N-tert-
butanesulfinyl imines was followed with 85.3 mg (0.366 mmol) of imine 6.40 and 2.0 mL (0.73
mmol) of freshly prepared benzyl zinc reagent 6.09 (0.37 M). Purification by flash
chromatography afforded separable diastereomeric products 6.41a (major) and 6.41b (minor) in
73:27 dr. 6.41a (RS,R,S): 70.0 mg, 59%, clear oil which became a white solid upon
concentration from CH2Cl2/CDCl3, m.p. 66.9–69.0 °C. IR (in CHCl3): 2986, 1372, 1216, 1057,
750 cm–1
. 1H NMR (400 MHz, CDCl3): δ 7.21 (apparent t, J = 7.2 Hz, 2H), 7.13 (apparent t, J =
8.3 Hz, 3H), 4.05 (td, J = 6.4, 3.3 Hz, 1H), 3.94–3.86 (m, 2H), 3.82 (dd, J = 8.6, 6.7 Hz, 1H),
3.51–3.42 (m, 1H), 2.82 (d, J = 7.4 Hz, 2H), 1.39 (s, 3H), 1.27 (s, 3H), 1.05 (s, 9H); 13
C NMR (126 MHz, CDCl3): δ 137.94, 129.72, 128.59, 126.69, 109.30, 76.39, 66.38, 57.95, 55.88, 40.86,
26.64, 25.21, 22.77. HRMS (m/z): [M+H]+ calcd. for C17H28NO3S, 326.1784; found, 326.1778.
6.41b (RS,S,S): 26.4 mg, 22%, white solid, m.p. 87.6–90.0 °C. IR (in CHCl3): 2983, 2926, 1370, 1214, 1065, 754 cm
–1.
1H NMR (400 MHz, CDCl3): δ 7.32–7.23 (m, 3H), 7.21–7.13 (m, 2H),
3.97–3.82 (m, 1H), 3.79–3.63 (m, 2H), 3.53–3.41 (m, 1H), 3.22 (d, J = 9.2 Hz, 1H), 3.14 (dd, J =
13.6, 4.1 Hz, 1H), 3.04 (dd, J = 13.6, 4.9 Hz, 1H), 1.40 (s, 3H), 1.30 (s, 3H), 1.07 (s, 9H); 13
C NMR (126 MHz, CDCl3): δ 135.63, 131.22, 128.67, 126.89, 109.84, 75.78, 67.96, 59.30, 56.29,
37.97, 27.06, 25.60, 22.72. HRMS (m/z): [M+H]+ calcd. for C17H28NO3S, 326.1784; found,
326.1778.
References
1. For reviews on tert-butanesulfinyl imine chemistry, see: (a) Robak, M. T.; Herbage, M. A.;
Ellman, J. A. Chem. Rev. 2010, 110, 3600; (b) Ferreira, F.; Botuha, C.; Chemla, F.; Perez-Luna,
A. Chem. Soc. Rev. 2009, 38, 1162.
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144
145
Appendix 6.1: X-ray crystal data for compound 6.27
146
Figure A6.1.1. X-ray crystal structure of 6.27 with thermal ellipsoids drawn at the 50% probability level.
Data collection parameters
A colorless needle crystal of SO4NC21H27 having approximate dimensions of 0.10 x 0.05 x 0.05
mm was mounted in a loop. All measurements were made on a Rigaku Saturn724 CCD
diffractometer using graphite monochromated Cu-K radiation.
The crystal-to-detector distance was 50.00 mm.
Cell constants and an orientation matrix for data collection corresponded to a primitive
orthorhombic cell with dimensions:
a = 5.57254(10) Å
b = 11.2226(2) Å
c = 32.524(2) Å
V = 2034.02(15) Å3
For Z = 4 and F.W. = 389.51, the calculated density is 1.272 g/cm3. The reflection conditions of:
h00: h = 2n
0k0: k = 2n
00l: l = 2n
uniquely determine the space group to be:
P212121 (#19)
147
The data were collected at a temperature of –180 ± 1 °C to a maximum 2 value of 131.8°. A
total of 321 oscillation images were collected. A sweep of data was done using scans from –
48.0 to 132.0° in 2.0° step, at = 45.0° and = 135.0°. The exposure rate was 15.0 [s/°]. The
detector swing angle was 42.00°. A second sweep was performed using scans from 0.0 to
180.0° in 2.0° step, at = 45.0° and = 135.0°. The exposure rate was 30.0 [s/°]. The detector
swing angle was 90.00°. Another sweep was performed using scans from 1.0 to 171.0° in 2.0°
step, at = 45.0° and = 0.0°. The exposure rate was 30.0 [s/°]. The detector swing angle was
90.00°. Another sweep was performed using scans from 98.0 to 150.0° in 2.0° step, at =
30.0° and = 0.0°. The exposure rate was 30.0 [s/°]. The detector swing angle was 90.00°.
Another sweep was performed using scans from 70.0 to 130.0° in 2.0° step, at = 30.0° and
= 180.0°. The exposure rate was 30.0 [s/°]. The detector swing angle was 90.00°. The crystal-to-
detector distance was 50.00 mm. Readout was performed in the 0.090 mm pixel mode.
Data reduction parameters
Of the 8763 reflections that were collected, 3362 were unique (Rint = 0.0689). Data were
collected and processed using CrystalClear (Rigaku).1
The linear absorption coefficient, , for Cu-K radiation is 16.260 cm–1
. An empirical
absorption correction was applied which resulted in transmission factors ranging from 0.513 to
0.922. The data were corrected for Lorentz and polarization effects.
Structure solution and refinement
The structure was solved by direct methods2 and expanded using Fourier techniques. The
non-hydrogen atoms were refined anisotropically. Hydrogen atoms were refined using the riding
model. The final cycle of full-matrix least-squares refinement3 on F
2 was based on 3351
observed reflections and 248 variable parameters and converged (largest parameter shift was
0.00 times its esd) with unweighted and weighted agreement factors of:
R1 = ||Fo| – |Fc|| / |Fo| = 0.0691
wR2 = [ ( w (Fo2 – Fc
2)2 )/ w (Fo
2)2]1/2
= 0.2016
The standard deviation of an observation of unit weight4 was 1.12. Unit weights were used.
The maximum and minimum peaks on the final difference Fourier map corresponded to 0.79 and
–0.37 e–/Å
3, respectively. The absolute structure was deduced based on Flack parameter, –
0.00(4), using 1309 Friedel pairs.5
Neutral atom scattering factors were taken from Cromer and Waber.6 Anomalous dispersion
effects were included in Fcalc;7 the values for f' and f" were those of Creagh and McAuley.
8
The values for the mass attenuation coefficients are those of Creagh and Hubbell.9 All
148
calculations were performed using the CrystalStructure10
crystallographic software package
except for refinement, which was performed using SHELXL-97.11
References
1. CrystalClear: Rigaku Corporation, 1999. CrystalClear Software User's Guide, Molecular
Structure Corporation, (c) 2000.J.W.Pflugrath (1999) Acta Cryst. D55, 1718–1725.
2. SIR2004: M.C. Burla, R. Caliandro, M. Camalli, B. Carrozzini, G.L. Cascarano, L. De Caro,
C. Giacovazzo, G. Polidori, R. Spagna (2005)
3. Least Squares function minimized: (SHELXL97)
w(Fo2–Fc
2)2 where w = Least Squares weights.
4. Standard deviation of an observation of unit weight:
[w(Fo2–Fc
2)2/(No–Nv)]
1/2 where No = number of observations, Nv = number of variables
5. Flack, H. D. (1983), Acta Cryst. A39, 876–881.
6. Cromer, D. T. & Waber, J. T.; "International Tables for X-ray Crystallography", Vol. IV, The
Kynoch Press, Birmingham, England, Table 2.2 A (1974).
7. Ibers, J. A. & Hamilton, W. C.; Acta Crystallogr., 17, 781 (1964).
8. Creagh, D. C. & McAuley, W.J .; "International Tables for Crystallography", Vol C, (A.J.C.
Wilson, ed.), Kluwer Academic Publishers, Boston, Table 4.2.6.8, pages 219–222 (1992).
9. Creagh, D. C. & Hubbell, J.H..; "International Tables for Crystallography", Vol C, (A.J.C.
Wilson, ed.), Kluwer Academic Publishers, Boston, Table 4.2.4.3, pages 200–206 (1992).
10. CrystalStructure 4.0: Crystal Structure Analysis Package, Rigaku and Rigaku Americas
(2000–2010). 9009 New Trails Dr. The Woodlands TX 77381 USA.
11. SHELX97: Sheldrick, G.M. (1997).
149
Table A6.1.1. Crystal data and structure refinement
Crystal data
Chemical formula SO4NC21H27
Mr 389.51
Crystal system, space group Orthorhombic, P212121
Temperature (K) 93
a, b, c (Å) 5.57254 (10), 11.2226 (2), 32.524 (2)
V (Å3) 2034.02 (15)
Z 4
Radiation type Cu Kα
µ (mm−1
) 1.63
Crystal size (mm) 0.10 × 0.05 × 0.05
Data collection
Diffractometer Rigaku Saturn724 CCD
diffractometer
Absorption correction Multi-scan
Jacobson, R. (1998) Private communication
Tmin, Tmax 0.513, 0.922
No. of measured, independent and
observed [F2 > 2.0σ(F
2)] reflections
8763, 3351, 2672
Rint 0.069
(sin θ/λ)max (Å−1
) 0.592
Refinementa
R[F2 > 2σ(F
2)], wR(F
2), S 0.069, 0.202, 1.12
No. of reflections 3351
No. of parameters 248
H-atom treatment H atoms treated by a mixture of independent and
constrained refinement
Δρmax, Δρmin (e Å−3
) 0.79, −0.37
Absolute structure Flack, H. D. (1983), Acta Cryst. A39, 876–881.
1309 Friedel Pairs
Absolute structure parameter −0.00 (4) aRefinement was performed using all reflections. The weighted R-factor (wR) and goodness of fit (S) are
based on F2. R-factor (gt) are based on F. The threshold expression of F
2 > 2.0 σ(F
2) is used only for
calculating R-factor (gt).
150
Table A6.1.2. Fractional atomic coordinates and equivalent isotropic displacement parameters (Å2)
atom x y z Beqa
S(1) 0.6429 (2) 0.96741 (13) 0.83005 (3) 4.73(3)
O(1) 0.4970 (9) 0.8597 (5) 0.82691 (12) 8.54(14)
O(2) 0.9719 (6) 0.4497 (3) 1.00033 (10) 4.77(7)
O(3) 1.3115 (5) 0.4525 (3) 0.96300 (9) 4.13(6)
O(4) 0.9617 (7) 1.5076 (3) 0.84357 (10) 5.25(8)
N(1) 0.8947 (7) 0.9472 (4) 0.85383 (11) 4.02(7)
C(1) 0.7607 (9) 0.9940 (5) 0.77831 (15) 4.87(10)
C(2) 0.9182 (14) 1.1049 (5) 0.7800 (2) 6.80(15)
C(3) 0.9074 (14) 0.8860 (6) 0.76333 (16) 6.26(14)
C(4) 0.5441 (12) 1.0116 (9) 0.75071 (19) 8.17(20)
C(5) 0.9023 (8) 0.9548 (4) 0.89899 (12) 3.58(7)
C(6) 0.9498 (8) 0.8356 (4) 0.91884 (13) 3.60(8)
C(7) 0.7955 (8) 0.7865 (4) 0.94808 (14) 3.89(8)
C(8) 0.8438 (8) 0.6776 (4) 0.96646 (12) 3.64(8)
C(9) 1.0483 (8) 0.6135 (4) 0.95581 (13) 3.58(8)
C(10) 1.2055 (8) 0.6616 (4) 0.92652 (13) 3.80(8)
C(11) 1.1569 (9) 0.7699 (4) 0.90809 (13) 3.90(8)
C(12) 1.1001 (8) 0.4983 (4) 0.97557 (13) 3.91(8)
C(13) 1.3799 (10) 0.3396 (4) 0.97945 (14) 4.25(8)
C(14) 1.0902 (8) 1.0464 (4) 0.91261 (14) 4.02(8)
C(15) 1.0521 (8) 1.1696 (4) 0.89535 (14) 3.94(8)
C(16) 1.2199 (9) 1.2224 (5) 0.86990 (15) 4.42(9)
C(17) 1.1876 (10) 1.3330 (5) 0.85314 (15) 4.54(10)
C(18) 0.9813 (10) 1.3971 (4) 0.86197 (14) 4.16(9)
C(19) 0.8101 (9) 1.3488 (4) 0.88779 (14) 4.17(9)
C(20) 0.8469 (9) 1.2356 (4) 0.90403 (14) 4.07(8)
C(21) 0.7524 (12) 1.5758 (5) 0.85211 (18) 5.67(12) a
Beq = 8/3 2(U11(aa*)2 + U22(bb*)
2 + U33(cc*)
2 + 2U12(aa*bb*)cos + 2U13(aa*cc*)cos + 2U23(bb*cc*)cos )
151
Table A6.1.3. Fractional atomic coordinates and isotropic displacement parameters for hydrogens (Å2)
atom x y z Biso
H(2A) 0.8254 1.1716 0.7912 8.16
H(2B) 0.9729 1.1248 0.7521 8.16
H(2C) 1.0574 1.0896 0.7976 8.16
H(3A) 0.9591 0.8993 0.7349 7.52
H(3B) 0.8078 0.8141 0.7646 7.52
H(3C) 1.0486 0.8757 0.7810 7.52
H(4A) 0.5978 1.0309 0.7228 9.80
H(4B) 0.4457 1.0770 0.7614 9.80
H(4C) 0.4488 0.9382 0.7501 9.80
H(5) 0.7419 0.9833 0.9086 4.30
H(7) 0.6542 0.8287 0.9556 4.66
H(8) 0.7365 0.6465 0.9865 4.37
H(10) 1.3469 0.6192 0.9193 4.56
H(11) 1.2640 0.8006 0.8880 4.69
H(13A) 1.3566 0.3399 1.0093 5.10
H(13B) 1.5492 0.3245 0.9732 5.10
H(13C) 1.2808 0.2769 0.9671 5.10
H(14A) 1.0889 1.0513 0.9430 4.82
H(14B) 1.2509 1.0179 0.9041 4.82
H(16) 1.3633 1.1802 0.8638 5.31
H(17) 1.3063 1.3658 0.8355 5.45
H(19) 0.6691 1.3924 0.8944 5.00
H(20) 0.7282 1.2024 0.9216 4.88
H(21A) 0.7431 1.5917 0.8817 6.81
H(21B) 0.6100 1.5312 0.8434 6.81
H(21C) 0.7600 1.6514 0.8371 6.81
H(1N) 1.028(11) 0.905(5) 0.8438(19) 5.8(14)
152
Table A6.1.4. Atomic displacement parameters (Å2)
a
atom U11
U22
U33
U12
U13
U23
S(1) 0.0537 (6) 0.0866 (8) 0.0395 (6) −0.0059 (6) −0.0022 (5) 0.0067 (6)
O(1) 0.120 (3) 0.154 (4) 0.051 (2) −0.084 (3) −0.017 (2) 0.015 (3)
O(2) 0.0638 (18) 0.0606 (18) 0.057 (2) −0.0011 (17) 0.0060 (17) 0.0111 (16)
O(3) 0.0551 (17) 0.0538 (16) 0.0478 (17) 0.0069 (14) 0.0050 (14) 0.0117 (14)
O(4) 0.099 (3) 0.0523 (19) 0.0477 (18) −0.0058 (18) −0.0026 (19) 0.0014 (14)
N(1) 0.054 (2) 0.065 (2) 0.0338 (18) 0.017 (2) −0.0050 (17) −0.0023 (16)
C(1) 0.058 (3) 0.083 (3) 0.043 (3) 0.000 (3) −0.007 (2) 0.005 (2)
C(2) 0.114 (5) 0.082 (4) 0.062 (3) −0.007 (4) 0.007 (4) 0.017 (3)
C(3) 0.106 (5) 0.089 (4) 0.043 (3) 0.001 (4) 0.008 (3) −0.015 (3)
C(4) 0.080 (4) 0.178 (8) 0.053 (3) 0.008 (5) −0.010 (3) 0.017 (4)
C(5) 0.050 (2) 0.054 (2) 0.032 (2) 0.005 (2) 0.0029 (19) −0.0027 (17)
C(6) 0.048 (2) 0.054 (2) 0.034 (2) 0.0011 (19) 0.000 (2) −0.0032 (18)
C(7) 0.048 (2) 0.059 (3) 0.040 (2) 0.0043 (19) 0.000 (2) −0.0014 (19)
C(8) 0.048 (2) 0.061 (2) 0.029 (2) −0.002 (2) 0.002 (2) 0.0007 (17)
C(9) 0.046 (2) 0.053 (2) 0.037 (2) −0.0030 (18) −0.0009 (19) 0.0045 (18)
C(10) 0.046 (2) 0.058 (3) 0.041 (2) −0.001 (2) 0.0080 (19) 0.0031 (19)
C(11) 0.051 (2) 0.057 (2) 0.040 (2) 0.001 (2) 0.005 (2) 0.0072 (19)
C(12) 0.050 (2) 0.059 (3) 0.040 (2) −0.004 (2) 0.002 (2) 0.0042 (19)
C(13) 0.065 (3) 0.051 (2) 0.045 (2) 0.005 (2) −0.004 (2) 0.0052 (19)
C(14) 0.053 (2) 0.055 (2) 0.044 (2) −0.001 (2) −0.003 (2) 0.003 (2)
C(15) 0.053 (2) 0.052 (2) 0.044 (2) 0.002 (2) −0.002 (2) 0.003 (2)
C(16) 0.057 (3) 0.063 (3) 0.048 (3) −0.001 (2) 0.001 (2) −0.002 (2)
C(17) 0.067 (3) 0.063 (3) 0.043 (3) −0.013 (2) 0.003 (2) −0.002 (2)
C(18) 0.069 (3) 0.048 (2) 0.041 (2) −0.007 (2) −0.005 (2) 0.0001 (19)
C(19) 0.057 (3) 0.056 (2) 0.045 (2) 0.006 (2) −0.009 (2) 0.002 (2)
C(20) 0.055 (2) 0.061 (3) 0.039 (2) 0.004 (2) −0.005 (2) 0.0044 (19)
C(21) 0.101 (4) 0.055 (3) 0.060 (3) 0.003 (3) −0.022 (3) 0.002 (2) a The general temperature factor expression:
exp(–22(a*2U11h
2 + b*
2U22k
2 + c*
2U33l
2 + 2a*b*U12hk + 2a*c*U13hl + 2b*c*U23kl))
153
Table A6.1.5. Bond lengths (Å)
bond length bond length
S(1)—O(1) 1.461 (6) N(1)—H(1N) 0.94 (6)
S(1)—N(1) 1.618 (4) C(2)—H(2A) 0.980
S(1)—C(1) 1.831 (5) C(2)—H(2B) 0.980
O(2)—C(12) 1.207 (5) C(2)—H(2C) 0.980
O(3)—C(12) 1.349 (5) C(3)—H(3A) 0.980
O(3)—C(13) 1.427 (5) C(3)—H(3B) 0.980
O(4)—C(18) 1.381 (6) C(3)—H(3C) 0.980
O(4)—C(21) 1.422 (7) C(4)—H(4A) 0.980
N(1)—C(5) 1.472 (5) C(4)—H(4B) 0.980
C(1)—C(2) 1.524 (9) C(4)—H(4C) 0.980
C(1)—C(3) 1.541 (8) C(5)—H(5) 1.000
C(1)—C(4) 1.517 (8) C(7)—H(7) 0.950
C(5)—C(6) 1.509 (6) C(8)—H(8) 0.950
C(5)—C(14) 1.533 (6) C(10)—H(10) 0.950
C(6)—C(7) 1.395 (6) C(11)—H(11) 0.950
C(6)—C(11) 1.414 (6) C(13)—H(13A) 0.980
C(7)—C(8) 1.387 (6) C(13)—H(13B) 0.980
C(8)—C(9) 1.391 (6) C(13)—H(13C) 0.980
C(9)—C(10) 1.402 (6) C(14)—H(14A) 0.990
C(9)—C(12) 1.473 (6) C(14)—H(14B) 0.990
C(10)—C(11) 1.382 (6) C(16)—H(16) 0.950
C(14)—C(15) 1.507 (6) C(17)—H(17) 0.950
C(15)—C(16) 1.382 (7) C(19)—H(19) 0.950
C(15)—C(20) 1.392 (7) C(20)—H(20) 0.950
C(16)—C(17) 1.367 (7) C(21)—H(21A) 0.980
C(17)—C(18) 1.386 (7) C(21)—H(21B) 0.980
C(18)—C(19) 1.382 (7) C(21)—H(21C) 0.980
C(19)—C(20) 1.391 (7)
154
Table A6.1.6. Bond angles (°)
bonds angle bonds angle
O(1)—S(1)—N(1) 113.6 (3) C(8)—C(9)—C(10) 118.8 (4)
O(1)—S(1)—C(1) 105.7 (2) C(8)—C(9)—C(12) 120.4 (4)
N(1)—S(1)—C(1) 98.7 (2) C(10)—C(9)—C(12) 120.8 (4)
C(12)—O(3)—C(13) 117.3 (4) C(9)—C(10)—C(11) 120.7 (4)
C(18)—O(4)—C(21) 117.6 (4) C(6)—C(11)—C(10) 120.7 (4)
S(1)—N(1)—C(5) 119.6 (3) O(2)—C(12)—O(3) 123.2 (4)
S(1)—C(1)—C(2) 107.9 (4) O(2)—C(12)—C(9) 124.9 (4)
S(1)—C(1)—C(3) 110.6 (4) O(3)—C(12)—C(9) 111.9 (4)
S(1)—C(1)—C(4) 106.2 (4) C(5)—C(14)—C(15) 114.3 (4)
C(2)—C(1)—C(3) 110.4 (5) C(14)—C(15)—C(16) 121.4 (4)
C(2)—C(1)—C(4) 111.9 (6) C(14)—C(15)—C(20) 121.9 (4)
C(3)—C(1)—C(4) 109.7 (5) C(16)—C(15)—C(20) 116.7 (4)
N(1)—C(5)—C(6) 112.4 (4) C(15)—C(16)—C(17) 122.6 (5)
N(1)—C(5)—C(14) 110.3 (3) C(16)—C(17)—C(18) 119.8 (5)
C(6)—C(5)—C(14) 110.6 (3) O(4)—C(18)—C(17) 116.2 (4)
C(5)—C(6)—C(7) 122.2 (4) O(4)—C(18)—C(19) 124.1 (5)
C(5)—C(6)—C(11) 120.0 (4) C(17)—C(18)—C(19) 119.7 (4)
C(7)—C(6)—C(11) 117.8 (4) C(18)—C(19)—C(20) 119.1 (4)
C(6)—C(7)—C(8) 121.5 (4) C(15)—C(20)—C(19) 122.0 (4)
C(7)—C(8)—C(9) 120.5 (4)
155
Table A6.1.7. Bond angles involving hydrogens (°)
bonds angle bonds angle
S(1)—N(1)—H(1N) 126 (4) C(11)—C(10)—H(10) 119.661
C(5)—N(1)—H(1N) 111 (4) C(6)—C(11)—H(11) 119.628
C(1)—C(2)—H(2A) 109.468 C(10)—C(11)—H(11) 119.626
C(1)—C(2)—H(2B) 109.47 O(3)—C(13)—H(13A) 109.471
C(1)—C(2)—H(2C) 109.47 O(3)—C(13)—H(13B) 109.466
H(2A)—C(2)—H(2B) 109.473 O(3)—C(13)—H(13C) 109.468
H(2A)—C(2)—H(2C) 109.475 H(13A)—C(13)—H(13B) 109.475
H(2B)—C(2)—H(2C) 109.471 H(13A)—C(13)—H(13C) 109.483
C(1)—C(3)—H(3A) 109.477 H(13B)—C(13)—H(13C) 109.464
C(1)—C(3)—H(3B) 109.466 C(5)—C(14)—H(14A) 108.688
C(1)—C(3)—H(3C) 109.457 C(5)—C(14)—H(14B) 108.685
H(3A)—C(3)—H(3B) 109.487 C(15)—C(14)—H(14A) 108.691
H(3A)—C(3)—H(3C) 109.483 C(15)—C(14)—H(14B) 108.686
H(3B)—C(3)—H(3C) 109.458 H(14A)—C(14)—H(14B) 107.615
C(1)—C(4)—H(4A) 109.473 C(15)—C(16)—H(16) 118.681
C(1)—C(4)—H(4B) 109.465 C(17)—C(16)—H(16) 118.675
C(1)—C(4)—H(4C) 109.475 C(16)—C(17)—H(17) 120.093
H(4A)—C(4)—H(4B) 109.48 C(18)—C(17)—H(17) 120.099
H(4A)—C(4)—H(4C) 109.467 C(18)—C(19)—H(19) 120.419
H(4B)—C(4)—H(4C) 109.467 C(20)—C(19)—H(19) 120.446
N(1)—C(5)—H(5) 107.801 C(15)—C(20)—H(20) 118.977
C(6)—C(5)—H(5) 107.804 C(19)—C(20)—H(20) 118.986
C(14)—C(5)—H(5) 107.801 O(4)—C(21)—H(21A) 109.47
C(6)—C(7)—H(7) 119.26 O(4)—C(21)—H(21B) 109.466
C(8)—C(7)—H(7) 119.287 O(4)—C(21)—H(21C) 109.474
C(7)—C(8)—H(8) 119.766 H(21A)—C(21)—H(21B) 109.474
C(9)—C(8)—H(8) 119.759 H(21A)—C(21)—H(21C) 109.475
C(9)—C(10)—H(10) 119.642 H(21B)—C(21)—H(21C) 109.469
156
Table A6.1.8. Torsion angles (°)a
bonds angle bonds angle
O(1)—S(1)—N(1)—C(5) −85.4 (3) C(11)—C(6)—C(7)—C(8) 0.7 (6)
O(1)—S(1)—C(1)—C(3) −58.3 (4) C(6)—C(7)—C(8)—C(9) −0.6 (6)
O(1)—S(1)—C(1)—C(4) 60.7 (4) C(7)—C(8)—C(9)—C(10) 0.6 (6)
N(1)—S(1)—C(1)—C(2) −61.5 (3) C(8)—C(9)—C(10)—C(11) −0.9 (6)
N(1)—S(1)—C(1)—C(3) 59.3 (3) C(8)—C(9)—C(12)—O(2) 2.8 (7)
C(13)—O(3)—C(12)—O(2) 1.6 (6) C(10)—C(9)—C(12)—O(3) 2.2 (6)
C(21)—O(4)—C(18)—C(19) 0.4 (6) C(9)—C(10)—C(11)—C(6) 1.0 (6)
S(1)—N(1)—C(5)—C(6) 111.3 (3) C(5)—C(14)—C(15)—C(16) −117.8 (4)
S(1)—N(1)—C(5)—C(14) −124.8 (3) C(5)—C(14)—C(15)—C(20) 62.0 (5)
N(1)—C(5)—C(6)—C(7) −123.9 (4) C(16)—C(15)—C(20)—C(19) 0.5 (7)
N(1)—C(5)—C(6)—C(11) 56.4 (5) C(20)—C(15)—C(16)—C(17) −1.2 (7)
N(1)—C(5)—C(14)—C(15) 57.0 (4) C(15)—C(16)—C(17)—C(18) 0.8 (7)
C(14)—C(5)—C(6)—C(7) 112.3 (4) C(16)—C(17)—C(18)—C(19) 0.2 (7)
C(14)—C(5)—C(6)—C(11) −67.4 (5) C(17)—C(18)—C(19)—C(20) −0.9 (7)
C(7)—C(6)—C(11)—C(10) −0.9 (6) C(18)—C(19)—C(20)—C(15) 0.5 (7) aThose having bond angles > 160 ° are excluded.
157
Appendix 6.2: X-ray crystal data for compound 6.31
158
Figure A6.2.1. X-ray crystal structure of 6.31 with thermal ellipsoids drawn at the 50% probability level.
Data collection parameters
A colorless plate crystal of SFONC16H26 having approximate dimensions of 0.18 x 0.10 x 0.08
mm was mounted in a loop. All measurements were made on a Rigaku R-AXIS RAPID imaging
plate diffractometer using graphite monochromated Cu-K radiation.
The crystal-to-detector distance was 127.40 mm.
Cell constants and an orientation matrix for data collection corresponded to a primitive
orthorhombic cell with dimensions:
a = 8.7454(2) Å
b = 10.5113(3) Å
c = 18.0667(13) Å
V = 1660.79(13) Å3
For Z = 4 and F.W. = 299.45, the calculated density is 1.198 g/cm3. The reflection conditions of:
h00: h = 2n
0k0: k = 2n
00l: l = 2n
uniquely determine the space group to be:
P212121 (#19)
159
The data were collected at a temperature of –180 + 1 °C to a maximum 2 value of 136.4°. A
total of 63 oscillation images were collected. A sweep of data was done using scans from 20.0
to 200.0° in 5.0° step, at = 54.0° and = 270.0°. The exposure rate was 48.0 [s/°]. A second
sweep was performed using scans from 20.0 to 125.0° in 5.0° step, at = 0.0° and = 90.0°.
The exposure rate was 48.0 [s/°]. Another sweep was performed using scans from 70.0 to
100.0° in 5.0° step, at = 54.0° and = 90.0°. The exposure rate was 48.0 [s/°]. The crystal-to-
detector distance was 127.40 mm. Readout was performed in the 0.100 mm pixel mode.
Data reduction parameters
Of the 6921 reflections that were collected, 2705 were unique (Rint = 0.0702).
The linear absorption coefficient, , for Cu-K radiation is 17.822 cm–1
. An empirical
absorption correction was applied which resulted in transmission factors ranging from 0.621 to
0.867. The data were corrected for Lorentz and polarization effects. A correction for secondary
extinction1 was applied (coefficient = 0.002850).
Structure solution and refinement
The structure was solved by direct methods2 and expanded using Fourier techniques. The
non-hydrogen atoms were refined anisotropically. Hydrogen atoms were refined using the riding
model. The final cycle of full-matrix least-squares refinement3 on F
2 was based on 2687
observed reflections and 186 variable parameters and converged (largest parameter shift was
0.00 times its esd) with unweighted and weighted agreement factors of:
R1 = ||Fo| – |Fc|| / |Fo| = 0.0769
wR2 = [ ( w (Fo2 – Fc
2)2 )/ w (Fo
2)2]1/2
= 0.2081
The standard deviation of an observation of unit weight4 was 1.12. Unit weights were used.
The maximum and minimum peaks on the final difference Fourier map corresponded to 0.77 and
–0.57 e–/Å
3, respectively. The absolute structure was deduced based on Flack parameter, 0.06(5),
using 959 Friedel pairs.5
Neutral atom scattering factors were taken from Cromer and Waber.6 Anomalous dispersion
effects were included in Fcalc;7 the values for f' and f" were those of Creagh and McAuley.
8
The values for the mass attenuation coefficients are those of Creagh and Hubbell.9 All
calculations were performed using the CrystalStructure10
crystallographic software package
except for refinement, which was performed using SHELXL-97.11
160
References
1. Larson, A.C. (1970), Crystallographic Computing, 291–294. F.R. Ahmed, ed. Munksgaard,
Copenhagen (equation 22, with V replaced by the cell volume).
2. SIR2004: M.C. Burla, R. Caliandro, M. Camalli, B. Carrozzini, G.L. Cascarano, L. De Caro,
C. Giacovazzo, G. Polidori, R. Spagna (2005)
3. Least Squares function minimized: (SHELXL97)
w(Fo2–Fc
2)2 where w = Least Squares weights.
4. Standard deviation of an observation of unit weight:
[w(Fo2–Fc
2)2/(No–Nv)]
1/2 where No = number of observations, Nv = number of variables
5. Flack, H. D. (1983), Acta Cryst. A39, 876–881.
6. Cromer, D. T. & Waber, J. T.; "International Tables for X-ray Crystallography", Vol. IV, The
Kynoch Press, Birmingham, England, Table 2.2 A (1974).
7. Ibers, J. A. & Hamilton, W. C.; Acta Crystallogr., 17, 781 (1964).
8. Creagh, D. C. & McAuley, W.J .; "International Tables for Crystallography", Vol C, (A.J.C.
Wilson, ed.), Kluwer Academic Publishers, Boston, Table 4.2.6.8, pages 219–222 (1992).
9. Creagh, D. C. & Hubbell, J.H..; "International Tables for Crystallography", Vol C, (A.J.C.
Wilson, ed.), Kluwer Academic Publishers, Boston, Table 4.2.4.3, pages 200–206 (1992).
10. CrystalStructure 4.0: Crystal Structure Analysis Package, Rigaku and Rigaku Americas
(2000–2010). 9009 New Trails Dr. The Woodlands TX 77381 USA.
11. SHELX97: Sheldrick, G.M. (1997).
161
Table A6.2.1. Crystal data and structure refinement
Crystal data
Chemical formula SFONC16H26
Mr 299.45
Crystal system, space group Orthorhombic, P212121
Temperature (K) 93
a, b, c (Å) 8.7454 (2), 10.5113 (3), 18.0667 (13)
V (Å3) 1660.79 (13)
Z 4
Radiation type Cu Kα
µ (mm−1
) 1.78
Crystal size (mm) 0.18 × 0.10 × 0.08
Data collection
Diffractometer Rigaku R-AXIS RAPID imaging plate
diffractometer
Absorption correction Multi-scan
Higashi, T. (1995). Program for Absorption
Correction. Rigaku Corporation, Tokyo, Japan.
Tmin, Tmax 0.621, 0.867
No. of measured, independent and
observed [F2 > 2.0σ(F
2)] reflections
6921, 2687, 1620
Rint 0.070
(sin θ/λ)max (Å−1
) 0.602
Refinementa
R[F2 > 2σ(F
2)], wR(F
2), S 0.077, 0.208, 1.12
No. of reflections 2687
No. of parameters 186
H-atom treatment H atoms treated by a mixture of independent and
constrained refinement
Δρmax, Δρmin (e Å−3
) 0.77, −0.57
Absolute structure Flack, H. D. (1983), Acta Cryst. A39, 876–881.
959 Friedel Pairs
Absolute structure parameter 0.06 (5) aRefinement was performed using all reflections. The weighted R-factor (wR) and goodness of fit (S) are
based on F2. R-factor (gt) are based on F. The threshold expression of F
2 > 2.0 σ(F
2) is used only for
calculating R-factor (gt).
162
Table A6.2.2. Fractional atomic coordinates and equivalent isotropic displacement parameters (Å2)
atom x y z Beqa
S(1) 0.47767 (18) 0.78195 (15) 0.38887 (9) 2.83(4)
F(1) 1.2008 (4) 0.8138 (3) 0.2114 (2) 3.70(9)
O(1) 0.3777 (4) 0.7220 (4) 0.4473 (2) 2.93(9)
N(1) 0.6157 (6) 0.8618 (5) 0.4287 (3) 2.59(11)
C(1) 0.5818 (7) 0.6484 (5) 0.3468 (3) 2.23(12)
C(2) 0.4592 (6) 0.5600 (6) 0.3162 (3) 3.33(14)
C(3) 0.6790 (7) 0.5800 (6) 0.4046 (3) 3.19(14)
C(4) 0.6763 (7) 0.7046 (6) 0.2831 (3) 3.06(13)
C(5) 0.6415 (7) 0.9996 (6) 0.4140 (3) 2.48(12)
C(6) 0.5310 (7) 1.0894 (6) 0.4567 (4) 2.80(13)
C(7) 0.3666 (7) 1.0567 (6) 0.4380 (4) 2.94(14)
C(8) 0.5575 (7) 1.0797 (6) 0.5409 (3) 3.37(14)
C(9) 0.5609 (7) 1.2271 (6) 0.4313 (4) 3.42(14)
C(10) 0.8133 (6) 1.0246 (6) 0.4267 (4) 2.81(13)
C(11) 0.9156 (7) 0.9652 (6) 0.3700 (4) 2.42(12)
C(12) 0.9228 (7) 1.0179 (6) 0.2987 (4) 2.84(13)
C(13) 1.0177 (8) 0.9661 (6) 0.2453 (4) 3.06(13)
C(14) 1.1021 (7) 0.8619 (6) 0.2639 (4) 2.80(13)
C(15) 1.1000 (7) 0.8069 (6) 0.3324 (4) 2.96(13)
C(16) 1.0032 (6) 0.8592 (5) 0.3860 (3) 2.60(12) a
Beq = 8/3 2(U11(aa*)2 + U22(bb*)
2 + U33(cc*)
2 + 2U12(aa*bb*)cos + 2U13(aa*cc*)cos + 2U23(bb*cc*)cos )
163
Table A6.2.3. Fractional atomic coordinates and isotropic displacement parameters for hydrogens (Å2)
atom x y z Biso
H(2A) 0.5077 0.4914 0.2879 3.99
H(2B) 0.3905 0.6082 0.2838 3.99
H(2C) 0.4004 0.5236 0.3572 3.99
H(3A) 0.7676 0.6328 0.4172 3.83
H(3B) 0.7141 0.4985 0.3845 3.83
H(3C) 0.6177 0.5649 0.4491 3.83
H(4A) 0.7325 0.6362 0.2581 3.67
H(4B) 0.7489 0.7670 0.3029 3.67
H(4C) 0.6079 0.7463 0.2477 3.67
H(5) 0.6218 1.0133 0.3601 2.98
H(7A) 0.2979 1.1169 0.4628 3.53
H(7B) 0.3439 0.9701 0.4549 3.53
H(7C) 0.3518 1.0621 0.3844 3.53
H(8A) 0.5347 0.9930 0.5575 4.05
H(8B) 0.4903 1.1399 0.5665 4.05
H(8C) 0.6643 1.1000 0.5522 4.05
H(9A) 0.6661 1.2512 0.4437 4.11
H(9B) 0.4895 1.2846 0.4563 4.11
H(9C) 0.5462 1.2331 0.3776 4.11
H(10A) 0.8310 1.1176 0.4269 3.37
H(10B) 0.8420 0.9917 0.4762 3.37
H(12) 0.8619 1.0900 0.2870 3.41
H(13) 1.0238 1.0021 0.1971 3.68
H(15) 1.1624 0.7353 0.3433 3.55
H(16) 0.9976 0.8216 0.4337 3.12
H(1N) 0.692 (6) 0.821 (5) 0.462 (3) 2.9(14)
164
Table A6.2.4. Atomic displacement parameters (Å2)
a
atom U11
U22
U33
U12
U13
U23
S(1) 0.0288 (8) 0.0319 (9) 0.0469 (10) 0.0007 (8) −0.0011 (8) 0.0002 (9)
F(1) 0.041 (2) 0.039 (2) 0.060 (3) 0.0005 (19) 0.021 (2) −0.003 (2)
O(1) 0.025 (2) 0.037 (3) 0.050 (3) −0.001 (2) 0.010 (2) 0.008 (2)
N(1) 0.026 (3) 0.029 (3) 0.043 (4) −0.005 (2) −0.010 (3) −0.002 (3)
C(1) 0.019 (3) 0.023 (3) 0.042 (4) −0.002 (3) 0.002 (3) −0.005 (3)
C(2) 0.029 (4) 0.036 (4) 0.062 (5) −0.002 (3) −0.007 (4) −0.004 (3)
C(3) 0.034 (4) 0.033 (4) 0.054 (5) 0.009 (3) −0.005 (4) 0.007 (4)
C(4) 0.035 (4) 0.038 (4) 0.043 (4) 0.004 (3) 0.005 (3) −0.002 (4)
C(5) 0.029 (3) 0.023 (3) 0.043 (4) −0.004 (3) 0.000 (3) 0.005 (3)
C(6) 0.033 (4) 0.031 (4) 0.042 (4) 0.003 (3) 0.001 (3) −0.001 (3)
C(7) 0.035 (4) 0.035 (4) 0.041 (4) 0.000 (3) 0.004 (3) −0.003 (3)
C(8) 0.042 (4) 0.047 (4) 0.040 (4) 0.000 (3) 0.001 (3) −0.001 (4)
C(9) 0.041 (4) 0.031 (4) 0.059 (5) 0.000 (3) 0.013 (3) 0.002 (4)
C(10) 0.027 (3) 0.026 (4) 0.054 (4) −0.009 (3) 0.000 (3) −0.003 (3)
C(11) 0.019 (3) 0.026 (3) 0.047 (4) 0.000 (3) −0.004 (3) −0.001 (3)
C(12) 0.028 (3) 0.032 (4) 0.048 (4) 0.000 (3) −0.002 (3) 0.011 (3)
C(13) 0.030 (4) 0.038 (4) 0.049 (4) 0.010 (3) 0.008 (3) 0.009 (3)
C(14) 0.026 (3) 0.035 (4) 0.045 (4) 0.002 (3) 0.011 (3) −0.003 (3)
C(15) 0.031 (3) 0.028 (4) 0.054 (4) −0.005 (3) −0.009 (3) 0.003 (3)
C(16) 0.027 (3) 0.030 (3) 0.042 (4) 0.004 (3) 0.001 (3) 0.006 (3) a The general temperature factor expression:
exp(–22(a*2U11h
2 + b*
2U22k
2 + c*
2U33l
2 + 2a*b*U12hk + 2a*c*U13hl + 2b*c*U23kl))
165
Table A6.2.5. Bond lengths (Å)
bond length bond length
S(1)—O(1) 1.508 (4) C(2)—H(2C) 0.980
S(1)—N(1) 1.637 (5) C(3)—H(3A) 0.980
S(1)—C(1) 1.838 (6) C(3)—H(3B) 0.980
F(1)—C(14) 1.378 (7) C(3)—H(3C) 0.980
N(1)—C(5) 1.489 (8) C(4)—H(4A) 0.980
C(1)—C(2) 1.523 (8) C(4)—H(4B) 0.980
C(1)—C(3) 1.526 (8) C(4)—H(4C) 0.980
C(1)—C(4) 1.534 (8) C(5)—H(5) 1.000
C(5)—C(6) 1.557 (9) C(7)—H(7A) 0.980
C(5)—C(10) 1.542 (8) C(7)—H(7B) 0.980
C(6)—C(7) 1.516 (9) C(7)—H(7C) 0.980
C(6)—C(8) 1.541 (8) C(8)—H(8A) 0.980
C(6)—C(9) 1.541 (9) C(8)—H(8B) 0.980
C(10)—C(11) 1.497 (8) C(8)—H(8C) 0.980
C(11)—C(12) 1.404 (9) C(9)—H(9A) 0.980
C(11)—C(16) 1.383 (8) C(9)—H(9B) 0.980
C(12)—C(13) 1.384 (9) C(9)—H(9C) 0.980
C(13)—C(14) 1.364 (9) C(10)—H(10A) 0.990
C(14)—C(15) 1.366 (9) C(10)—H(10B) 0.990
C(15)—C(16) 1.398 (9) C(12)—H(12) 0.950
N(1)—H(1N) 1.00 (5) C(13)—H(13) 0.950
C(2)—H(2A) 0.980 C(15)—H(15) 0.950
C(2)—H(2B) 0.980 C(16)—H(16) 0.950
166
Table A6.2.6. Bond angles (°)
bonds angle bonds angle
O(1)—S(1)—N(1) 109.5 (3) C(5)—C(6)—C(9) 108.5 (5)
O(1)—S(1)—C(1) 104.9 (3) C(7)—C(6)—C(8) 110.3 (5)
N(1)—S(1)—C(1) 102.0 (3) C(7)—C(6)—C(9) 107.9 (5)
S(1)—N(1)—C(5) 122.2 (4) C(8)—C(6)—C(9) 109.4 (5)
S(1)—C(1)—C(2) 105.5 (4) C(5)—C(10)—C(11) 114.1 (5)
S(1)—C(1)—C(3) 110.6 (4) C(10)—C(11)—C(12) 119.4 (5)
S(1)—C(1)—C(4) 106.4 (4) C(10)—C(11)—C(16) 121.6 (6)
C(2)—C(1)—C(3) 110.7 (5) C(12)—C(11)—C(16) 119.0 (6)
C(2)—C(1)—C(4) 110.0 (5) C(11)—C(12)—C(13) 120.8 (6)
C(3)—C(1)—C(4) 113.2 (5) C(12)—C(13)—C(14) 117.9 (6)
N(1)—C(5)—C(6) 114.0 (5) F(1)—C(14)—C(13) 117.6 (6)
N(1)—C(5)—C(10) 106.7 (5) F(1)—C(14)—C(15) 118.5 (5)
C(6)—C(5)—C(10) 115.3 (5) C(13)—C(14)—C(15) 123.9 (6)
C(5)—C(6)—C(7) 109.9 (5) C(14)—C(15)—C(16) 117.9 (6)
C(5)—C(6)—C(8) 110.8 (5) C(11)—C(16)—C(15) 120.5 (6)
167
Table A6.2.7. Bond angles involving hydrogens (°)
bonds angle bonds angle
S(1)—N(1)—H(1N) 122 (3) H(7A)—C(7)—H(7C) 109.473
C(5)—N(1)—H(1N) 115 (3) H(7B)—C(7)—H(7C) 109.477
C(1)—C(2)—H(2A) 109.468 C(6)—C(8)—H(8A) 109.464
C(1)—C(2)—H(2B) 109.473 C(6)—C(8)—H(8B) 109.473
C(1)—C(2)—H(2C) 109.478 C(6)—C(8)—H(8C) 109.469
H(2A)—C(2)—H(2B) 109.469 H(8A)—C(8)—H(8B) 109.475
H(2A)—C(2)—H(2C) 109.467 H(8A)—C(8)—H(8C) 109.475
H(2B)—C(2)—H(2C) 109.472 H(8B)—C(8)—H(8C) 109.472
C(1)—C(3)—H(3A) 109.471 C(6)—C(9)—H(9A) 109.472
C(1)—C(3)—H(3B) 109.468 C(6)—C(9)—H(9B) 109.475
C(1)—C(3)—H(3C) 109.475 C(6)—C(9)—H(9C) 109.471
H(3A)—C(3)—H(3B) 109.469 H(9A)—C(9)—H(9B) 109.469
H(3A)—C(3)—H(3C) 109.47 H(9A)—C(9)—H(9C) 109.464
H(3B)—C(3)—H(3C) 109.474 H(9B)—C(9)—H(9C) 109.475
C(1)—C(4)—H(4A) 109.472 C(5)—C(10)—H(10A) 108.726
C(1)—C(4)—H(4B) 109.479 C(5)—C(10)—H(10B) 108.727
C(1)—C(4)—H(4C) 109.475 C(11)—C(10)—H(10A) 108.718
H(4A)—C(4)—H(4B) 109.465 C(11)—C(10)—H(10B) 108.726
H(4A)—C(4)—H(4C) 109.467 H(10A)—C(10)—H(10B) 107.642
H(4B)—C(4)—H(4C) 109.469 C(11)—C(12)—H(12) 119.62
N(1)—C(5)—H(5) 106.749 C(13)—C(12)—H(12) 119.628
C(6)—C(5)—H(5) 106.751 C(12)—C(13)—H(13) 121.035
C(10)—C(5)—H(5) 106.753 C(14)—C(13)—H(13) 121.045
C(6)—C(7)—H(7A) 109.47 C(14)—C(15)—H(15) 121.037
C(6)—C(7)—H(7B) 109.47 C(16)—C(15)—H(15) 121.041
C(6)—C(7)—H(7C) 109.468 C(11)—C(16)—H(16) 119.743
H(7A)—C(7)—H(7B) 109.47 C(15)—C(16)—H(16) 119.733
168
Table A6.2.8. Torsion angles (°)a
bonds angle bonds angle
O(1)—S(1)—N(1)—C(5) −123.6 (4) C(10)—C(5)—C(6)—C(8) −59.1 (6)
O(1)—S(1)—C(1)—C(2) 59.1 (4) C(10)—C(5)—C(6)—C(9) 61.0 (6)
O(1)—S(1)—C(1)—C(3) −60.6 (4) C(5)—C(10)—C(11)—C(12) 73.5 (6)
N(1)—S(1)—C(1)—C(3) 53.6 (4) C(5)—C(10)—C(11)—C(16) −106.4 (6)
N(1)—S(1)—C(1)—C(4) −69.8 (4) C(12)—C(11)—C(16)—C(15) 1.3 (8)
C(1)—S(1)—N(1)—C(5) 125.7 (4) C(16)—C(11)—C(12)—C(13) −0.9 (8)
S(1)—N(1)—C(5)—C(6) 79.2 (5) C(11)—C(12)—C(13)—C(14) 0.7 (9)
S(1)—N(1)—C(5)—C(10) −152.3 (3) C(12)—C(13)—C(14)—C(15) −1.0 (9)
N(1)—C(5)—C(6)—C(7) −57.3 (6) C(13)—C(14)—C(15)—C(16) 1.4 (9)
N(1)—C(5)—C(6)—C(8) 64.9 (6) C(14)—C(15)—C(16)—C(11) −1.5 (8)
N(1)—C(5)—C(10)—C(11) 69.2 (6) aThose having bond angles > 160 ° are excluded.